Methods for transforming Phaffia strains, transformed Phaffia strains so obtained and recombinant DNA in said methods

ABSTRACT

The present invention provides recombinant DNA comprising a transcription promoter and a downstream sequence to be expressed, in operable linkage therewith, wherein the transcription promoter comprises a region found upstream of the open reading frame of a highly expressed Phaffia gene, preferably a glycolytic pathway gene, more preferably the gene coding for Glyceraldehyde-3-Phosphate Dehydrogenase. Further preferred recombinant DNAs according to the invention contain promoters of ribosomal protein encoding genes, more preferably wherein the transcription promoter comprises a region found upstream of the open reading frame encoding a protein as represented by one of the disclosed amino acid sequences. According to a further aspect of the invention an isolated DNA sequence coding for an enzyme involved in the carotenoid biosynthetic pathway of  Phaffia rhodozyma  is provided.

TECHNICAL FIELD

The present invention relates to methods for transforming Phaffia yeast, transformed Phaffia strains, as well as recombinant DNA for use therein.

BACKGROUND OF THE INVENTION

Methods for transforming the yeast Phaffia rhodozyma have been disclosed in European patent application 0 590 707 A1. These methods involve incubation of protoplasts with DNA or incubation of Phaffia cells with DNA followed by lithium acetate treatment. The recombinant DNA used to transform Phaffia strains with either of these methods comprised a Phaffia actin gene promoter to drive expression of the selectable marker genes coding for resistance against G418 or phleomycin. The methods involve long PEG and lithium acetate incubation times and transformation frequencies are low. When protoplasts are used, the transformation frequency is dependent on the quality of the protoplast suspension, making the procedure less reliable.

Recently a method for transforming Phaffia strains has been reported by Adrio J. L. and Veiga M. (July 1995, Biotechnology Techniques Vol. 9, No. 7, pp. 509-512). With this method the w transformation frequencies are in the range of 3 to 13 transformants per μg DNA, which is low. A further disadvantage of the method disclosed by these authors consists in increased doubling time of the transformed cells. The authors hypothesised that this may be due to interference of the autonomously replicating vector with chromosome replication.

Clearly, there is still a need for a reliable and efficient method of transforming Phaffia strains with foreign DNA. It is an objective of the present invention to provide methods and means to achieve this. It is a further objective of the invention to optimize expression of certain genes in Phaffia rhodozyma in order to make Phaffia a more suitable production host for certain valuable compounds.

SUMMARY OF THE INVENTION

The invention provides a method for obtaining a transformed Phaffia strain, comprising the steps of contacting cells or protoplasts of a Phaffia strain with recombinant DNA under conditions conducive to uptake thereof, said recombinant DNA comprising a transcription promoter and a downstream sequence to be expressed which is heterologous to said transcription promoter, in operable linkage therewith, identifying Phaffia rhodozyma cells or protoplasts having obtained the said recombinant DNA in expressible form, wherein the transcription promoter comprises a region that is found upstream of the open reading frame of a highly expressed Phaffia gene. According to a preferred embodiment of the invention said highly expressed Phaffia gene is a glycolytic pathway gene, more preferably the glycolytic pathway gene is coding for Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH). According to one aspect of the invention, said heterologous downstream sequence comprises an open reading frame coding for resistance against a selective agent, such as G418 or phleomycin.

Another preferred method according to the invention is one, wherein said recombinant DNA comprises further a transcription terminator downstream from the said DNA to be expressed, in operable linkage therewith, which transcription terminator comprises a region found downstream of the open reading frame of a Phaffia gene. It is still further preferred, that the recombinant DNA is in the form of linear DNA.

Another preferred embodiment comprises, in addition to the steps above, the step of providing an electropulse after contacting of Phaffia cells or protoplasts with DNA.

According to another embodiment the invention provides a transformed Phaffia strain capable of high-level expression of a heterologous DNA sequence, which strain is obtainable by a method according to the invention. Preferably, said Phaffia strain contains at least 10 copies of the said recombinant DNA integrated into its genome, such as a chromosome, particularly in the ribosomal DNA locus of said chromosome.

The invention also provides recombinant DNA comprising a transcription promoter and a heterologous downstream sequence to be expressed, in operable linkage therewith, wherein the transcription promoter comprises a region found upstream of the open reading frame of a highly expressed Phyla gene, preferably a glycolytic pathway gene, more preferably a gene coding for Glyceraldehyde-3-Phosphate Dehydrogenase.

Also provided is recombinant DNA according to the invention, wherein the heterologous downstream sequence comprises an open reading frame coding for reduced sensitivity against a selective agent, preferably G418 or phleomycin. Said recombinant DNA preferably comprises further a transcription terminator downstream from the said heterologous DNA sequence to be expressed, in operable linkage therewith.

Further aspects of the invention concern a microorganism harbouring recombinant DNA according to the invention, preferably Phaffia strains, more preferably Phaffia rhodozyma strains, as well as cultures thereof.

According to still other preferred embodiments isolated DNA fragments are provided comprising a Phaffia GAPDH-gene, or a fragment thereof, as well as the use of such a fragment for making a recombinant DNA construct. According to one embodiment of this aspect said fragment is a regulatory region located upstream or downstream of the open reading frame coding for GAPDH, and it is used in conjunction with a heterologous sequence to be expressed under the control thereof

The invention according to yet another aspect, provides a method for producing a protein or a pigment by culturing a Phaffia strain under conditions conducive to the production of said protein or pigment, wherein the Phaffia strain is a transformed Phaffia strain according to the invention.

According to another aspect of the invention, a method for obtaining a transformed Phaffia strain, comprising the steps of

contacting cells or protoplasts of a Phaffia strain with recombinant DNA under conditions conducive to uptake thereof,

said recombinant DNA comprising a transcription promoter and a downstream sequence to be expressed in operable linkage therewith,

identifying Phaffia rhodozyma cells or protoplasts having obtained the said recombinant DNA in expressible form,

wherein the downstream am sequence to be expressed comprise s an isolate d DNA sequence coding for an enzyme involved in the carotenoid biosynthetic pathway of Phaffia rhodozyma. Preferably, said enzyme has an activity selected from geranylgeranyl pyrophosphate synthase (crtE), phytoene synthase (crtB), phytoene desaturase (crtI) and lycopene cyclase (crtY), more preferably an enzyme having an amino acid sequence selected from the one represented by SEQIDNO: 13, SEQIDNO: 15, SEQIDNO: 17 and SEQIDNO: 19. According to a further embodiment, the transcription promoter is heterologous to said isolated DNA sequence, such as a glycolytic pathway gene in Phaffia. Especially preferred according to this embodiment is the Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) gene promoter.

Also provided is a transformed Phaffia strain obtainable by a method according to the invention and capable of expressing preferably over-expressing the DNA sequence encoding an enzyme, involved in the carotenoid biosynthesis pathway gene.

The invention is also embodied in recombinant DNA comprising an isolated DNA sequence according to the invention, preferably in the form of a vector.

Also claimed is the use of such a vector to transform a host, such as a Phaffia strain:

A host obtainable by transformation, optionally of an ancestor, using a method according to any one of claims 1 to 5, wherein said host is preferably capable of over-expressing DNA according to the invention.

According to a further embodiment a method is provided for expressing an enzyme involved in the carotenoid biosynthesis pathway, by culturing a host according to the invention under conditions conducive to the production of said enzyme. Also provided is a method for producing a carotenoid by cultivating a host according to the invention under conditions conducive to the production of carotenoid.

The following figures further illustrate the invention.

DESCRIPTION OF THE FIGURES

FIGS. 1A-1C Mapping of the restriction sites around the Phaffia rhodozyma GAPDH gene. Ethidium bromide stained 0.8% agarose gel (A) and Southern blot of chromosomal DNA (B) and cosmid pPRGDHcos1 (C) digested with several restriction enzymes and hybridized with the 300-bp PCR fragment of the, Phaffia rhodozyma GAPDH gene. Lane 1, DNA xKpnI; 2, xPstI; 3, xSmaI; 4, xSphI; L, lambda DNA digested with BstEII; 5, xSstI; 6, xXbaI and 7, xXhoI. The blot was hybridized in 6×SSC, 5×Denhardt's, 0.1% SDS, 100 ng/ml herring sperm DNA at 65° C. and washed with 0.1×SSC/0.1% SDS at 65° C. Exposure time of the autoradiogram was 16 h for the cosmid and 48 h from the blot containing the chromosomal DNA.

FIGS. 2A and 2B. The organisation of two subclones; pPRGDH3 and derivative (A) and pPRGDH6 and derivatives (B) containing (a part of) the GAPDH gene of Phaffia rhodozyma. The PCR probe is indicated by a solid box. The direction and extent of the sequence determination is indicated by arrows. solid boxes: GAPDH4 coding sequence open box: 5′ upstream and promoter region of GAPDH open box: 3′ non-coding Phaffia rhodozyma GAPDH sequence solid line: GAPDH intron hatched box: Poly-linker containing sites for different restriction enzymes dotted line: deleted fragments

FIG. 3. Cloning diagram of Phaffia transformation vector; pPR2. solid box: 5′ upstream and promoter sequence of GAPDH hatched box: G418 solid line: pUC19 open box: ribosomal DNA of Phaffia rhodozyma Only restriction sites used for cloning are indicated.

FIG. 4. Construction of pPR2T from pPR2T. Solid box (BamHI-HindIII fragment): GAPDH transcription terminator from Phaffia. All other boxes and lines arc as in FIG. 3. Only relevant details have been depicted.

FIG. 5. Detailed physical map of pGB-Ph9. bps=basepairs; rDNA ribosomal DNA locus of Phaffia; act.pro 2=actin transcription promoter; act.1 5′ non-translated and aminoterminal region of the open reading frame; NON COD.=non-coding region downstream of G418-gene;

FIG. 6. Detailed physical map of pPR2. GPDHpro=GAPDH transcription promoter region from Phaffia. Other acronyms as in FIG. 5.

FIG. 7. Detailed physical map of pPR2T. Tgdh=GAPDH transcription terminator of Phaffia. All other acronyms as in FIGS. 5 and 6.

FIG. 8. Overview of the carotenoid biosynthetic pathway of Erwinia uredovora.

FIGS. 9a-9 c. Representation of cDNA fragments and a restriction enzyme map of the plasmids pPRcrtE (A); pPRcrtB (B), pPRcrtI (C) and pPRcrtY (B).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides in generalised terms a method for obtaining a transformed Phaffia strain, comprising the steps of

contacting cells or protoplasts of a Phaffia strain with recombinant DNA under conditions conducive to uptake thereof,

said recombinant DNA comprising a transcription promoter and a downstream sequence to be expressed which is heterologous to said transcription promoter, in operable linkage therewith,

identifying Phaffia rhodozyma cells or protoplasts having obtained the said recombinant DNA in expressible form,

wherein the transcription promoter comprises a region that is found upstream of the open reading frame of a highly expressed Phaffia gene.

In order to illustrate the various ways of practicing the invention, some embodiments will be high-lighted and the meaning or scope of certain phrases will be elucidated.

The meaning of the expression recombinant DNA is well known in the art of genetic modification, meaning that a DNA molecule is provided, single or double stranded, either linear or circular, nicked or otherwise, characterised by the joining of at least two fragments of different origin. Such joining is usually, but not necessarily done in vitro. Thus, within the ambit of the claim are molecules which comprise DNA from different organisms or different genes of the same organism, or even different regions of the same gene, provided the regions are not adjacent in nature. The recombinant DNA according to the invention is characterised by a transcription promoter found upstream of an open reading frame of a highly expressed Phaffia gene, fused to a heterologous DNA sequence. With heterologous is meant ‘not naturally adjacent’. Thus the heterologous DNA sequence may be from a different organisms, a different gene from the same organism, or even of the same gene as the promoter, provided that the downstream sequence has been modified, usually in vitro. Such modification may be an insertion, deletion or substitution, affecting the encoded protein and/or its entrance into the secretory pathway, and/or its post-translational processing, and/or its codon usage.

The strong transcription promoter according to the invention must be in operable linkage with the heterologous downstream sequence in order to allow the transcriptional and translational machinery to recognise the starting signals. The regions upstream of open reading frames of highly expressed Phaffia genes contain TATA-like structures which are positioned at 26 to about 40 nucleotides upstream of the cap-site; the latter roughly corresponds with the transcriptional start site. Thus in order to allow transcription of the heterologous downstream sequence to start at the right location similar distances are to be respected. It is common knowledge, however, that there is a certain tolerance in the location of the TATA-signal relative to the transcription start site. Typically, mRNAs of the eukaryotic type contain a 5′-untranslated leader sequence (5′-utl), which is the region spanning the transcription start site to the start of translation; this region may vary from 30 to more than 200 nucleotides. Neither the length nor the origin of the 5′-utl is very critical; preferably it will be between 30 and 200 nucleotides. It may be from the same gene as the promoter, or it may be from the gene coding for the heterologous protein. It is well known that eukaryotic genes contain signals for the termination of transcription and/or polyadenylation, downstream of the open reading frame. The location of the termination signal is variable, but will typically be between 10 and 200 nucleotides downstream from the translational stop site (the end of the open reading frame), more usually between 30 and 100 nucleotides downstream from the translational stop site. Although the choice of the transcription terminator is not critical, it is found, that the when the terminator is selected from a region downstream of a Phaffia gene, preferably of a highly expressed Phaffia gene, more preferably from the GAPDH-encoding gene, the level of expression, as well as the frequency of transformation is improved.

It was found that significant numbers of clones were obtained which could grow on very high G418 concentrations (up to, and over, 1 mg/ml). Transcription promoters according to the invention are said to be from highly expressed genes, when they can serve to allow growth of transformed Phaffia cells, when linked to a G418 resistance gene as disclosed in the Examples, in the presence of at least 200 μm/ml, preferably more than 400, even more preferably more than 600, still more preferably more than 800 μg/ml of G418 in the growth medium. Examples of such promoters are, in addition to the promoter upstream from the GAPDH-gene in Phaffia, the promoters from Phaffia genes which are homologous to highly expressed genes from other yeasts, such as Pichia, Saccharomyces, Kluyveromyces, or fungi, such as Trichoderma, Aspergillus, and the like. Promoters which fulfill the requirements according to the invention, may be isolated from genomic DNA using molecular biological techniques which are, as such, all available to the person skilled in the art. The present invention provides a novel strategy for isolating strong promoters from Phaffia as follows. A cDNA-library is made from Phaffia mRNA, using known methods. Then for a number of clones with a cDNA insert, the DNA fragment (which represents the cDNA complement of the expressed mRNA) is sequenced. As a rule all fragments represent expressed genes from Phaffia. Moreover, genes that are abundantly expressed (such as the glycolytic promoters) are overrepresented in the mRNA population. Thus, the number of DNA-fragments to be sequenced in order to find a highly expressed gene, is limited to less than 100, probably even less than 50. The sequencing as such is routine, and should not take more than a couple of weeks. The nucleotide sequences obtained from this limited number of fragments, is subsequently compared to the known sequences stored in electronic databases such as EMBL or Geneseq. If a fragment shows homology of more than 50% over a given length (preferably more than 100 basepairs) the fragment is likely to represent the Phaffia equivalent of the gene found in the electronic database. In yeasts other than Phaffia, a number of highly expressed genes have been identified. These genes include the glycolytic pathway genes, phosphoglucoisomerase, phosphofructokinase, phosphotrioseisomerase, phosphoglucomutase, enolase, pyruvate kinase, alcohol dehydrogenase genes (EP 120 551, EP 0 164 556; Rosenberg S. et al., 1990, Meth. Enzymol.: 185, 341-351; Tuite M. F. 1982, EMBO J. 1, 603-608; Price V. et al., 1990, Meth. Enzymol. 185, 308-318) and the galactose regulon (Johnston, S. A. et al., 1987, Cell 50, 143-146). Accordingly, those Phaffia cDNA fragments that are significantly homologous to the highly expressed yeast genes (more than 40%, preferably more than 50% identity in a best match comparison over a range of more than 50, preferably more than 100 nucleotides) should be used to screen a genomic library from Phaffia, to find the corresponding gene. Employing this method, 14 higly expressed mRNAs from Phaffia rhodozyma have been copied into DNA, sequenced, and their (putative), open reading frames compared to a nucleic acid and amino amino acid sequence databases. It turned out that 13 out of these fourteen cDNAs coded for ribosomal protein genes, of which one coded simultaneously to ubiquitin; one cDNA codes for a glucose-repressed gene. The isolation of the genes and the promoters usually found upstream of the coding regions of these genes is now underway, and it is anticipated that each of these transcription promoters may advantageously be used to express heterologous genes, such as carotenoid biosynthesis genes. Among the genes and transcription promoters especially preferred according to this invention are the promoter found upstream of the ubiquitin-ribosomal 40S protein corresponding to the cDNA represented in SEQIDNO:10, the glucose-repressed cDNA represented in SEQIDNO:26, the 40S ribosomal protein S27 encoding cDNA represented in SEQIDNO:28, the 60S ribosomal protein Pla encoding cDNA represented by SEQIDNO:30, the 60S ribosomal protein L37e encoding cDNA represented in SEQIDNO:32, the 60S ribosomal protein L27a encoding cDNA represented in SEQIDNO:34, the 60S ribosomal protein L25 encoding cDNA represented in SEQIDNO:36, the 60S ribosomal protein P2 encoding cDNA represented in SEQIDNO:38, the 40S ribosomal protein S17A/B encoding cDNA represented in SEQIDNO:40, the 40S ribosomal protein S31 encoding cDNA represented in SEQIDNO:42, the 40S ribosomal protein S10 encoding cDNA represented in SEQIDNO:44, the 60S ribosomal protein L37A encoding cDNA represented in SEQIDNO:46, the 60S ribosomal protein L34 encoding cDNA represented in SEQIDNO:48, or the 40S ribosomal protein S16 encoding cDNA represented in SEQIDNO:50.

Promoters from these or other highly expressed genes can be picked up by the method according to the invention using only routine skills of (a) making a cDNA library on mRNA isolated from a Phaffia strain grown under desired conditions, (b) determining (part of) the nucleotide sequence of the (partial) cDNAs obtained in step (a), (c) comparing the obtained sequence data in step (b) to known sequence data, such as that stored in electronic databases, (d) cloning putative promoter fragments of the gene located either directly upstream of the open reading frame or directly upstream of the transcription start site of the gene corresponding to the expressed cDNA, and (e) verifying whether promoter sequences have been obtained by expressing a suitable marker, such as the G418 resistance gene, or a suitable non-selectable “reporter” sequence downstream from a fragment obtained in (d), transforming the DNA into a Phaffia rhodozyma strain and determining the level of expression of the marker gene or reporter sequence of transformants. A transcriptional promoter is said to be of a highly expressed gene if it is capable of making Phaffia rhodozyma cells transformed with a DNA construct comprising the said promoter linked uptream of the G418 resistance marker resistant to G418 in concentrations exceeding 200 μg per liter culture medium, preferably at least 400, more prefereably more than 600 μg/l. Especially preferred promoters are those conferring resistance against more than 800 μg/ml G418 in the growth medium.

Optionally, the transcriptional start site may be determined of the gene corresponding to the cDNA corresponding to a highly expressed gene, prior to cloning the putative promoter sequences; this may serve to locate the transcriptional initiation site more precisely, and moreover, helps to determine the length of the 5′-non-translated leader of the gene. To determine the location of the transcription start site, reverse primer extension, or classical S1-mapping may be performed, based on the knowledge of the cDNA sequence. Thus the exact location of the transcription promoter can be determined without undue burden, and the isolation of a fragment upstream of the transcription start site and containing the promoter, from a hybridising genomic clone (for example a phage or cosmid) is routine. Cloning the putative promoter fragment in front (upstream) of the coding region of, for example the G418-resistance gene, and transforming the gene cassette to Phaffia in order to evaluate the level of G418 resistance, and hence the level of expression of the G418-resistance gene as a consequence of the presence of the promoter is routine.

In a manner essentially as described for the isolation of other strong promoters, above, a transcription terminator may be isolated, with the proviso, that the terminator is located downstream from the open reading frame. The transcription stop site can be determined using procedures which are essentially the same as for the determination of the transcription start site. All these procedures are well known to those of skill in the art. A useful handbook is Nucleic Acid Hybridisation, Edited by B. D. Hames & S. J. Higgins, IRL Press Ltd., 1985; or Sambrook, sub. However, it is not critical that the transcription terminator is isolated from a highly expressed Phaffia gene, as long as it is from an expressed gene.

Using recombinant DNA according to the invention wherein the open reading frame codes for reduced sensitivity against G418, a transformation frequency was obtained up to 160 transformants per μg of linear DNA, at a G418 concentration in the medium of 40 μg/ml.

About 10 to 20 times as much transformed colonies were obtained with the vector according to the invention (pPR2) than with the prior art vector pGB-Ph9, disclosed in EP 0 590 707 A1 ( see Table 2; in the experiment of Example 7, the improvement is even more striking).

The method according to the invention calls for conditions conducive to uptake of the recombinant DNA. Such conditions have been disclosed in EP 509 707. They include but are not limited to the preparation of protoplasts using standard procedures known to those of skill in the art, and subsequent incubation with the recombinant DNA. Alternatively, Phaffia cells may be incubated overnight in the presence of LiAc and recombinant DNA. Still further alternative methods involve the use of particle acceleration. According to a preferred embodiment, the conditions conducive to uptake involve electroporation of recombinant DNA into Phaffia cells, such as described by Faber et al., (1994, Current Genetics 25, 305-310). Especially preferred conditions comprise electroporation, wherein the recombinant DNA comprises Phaffia ribosomal DNA, said recombinant DNA being in the linear form, most preferably by cleaving said recombinant DNA in the said ribosomal region. Still further preferred conditions, comprise the use of recombinant DNA in amounts of between 1 and 10 μg per 10⁸ cells, more preferably about 5 μg recombinant DNA is used per 2×10⁸ cells, which are cultivated for 16 h at 21° C.

Once cells have been transformed according to the method, identification of transformed cells may take place using any suitable technique. Thus, identification may be done by hybridisation techniques, DNA amplification techniques such a polymerase chain reaction using primers based on the recombinant DNA used, and the like. A preferred method of identifying transformed cells is one which employs selection for the recombinant DNA that comprises a gene coding for reduced sensitivity against a selective agent. A useful selective agent is G418, hygromycin, phleomycin and amdS. Genes that code for reduced sensitivity against these selective agents are well known in the art. The open reading frames of these genes may be used as the heterologous downstream sequence according to the invention, allowing selective enrichment of transformed cells, prior to identification of transformed cells. Once transformed cells have been identified they may used for further manipulation, or used directly in the production of valuable compounds, preferably in large scale fermentors.

It will be clear, that a very efficient method for transforming Phaffia strains has been disclosed. Moreover, not only the frequency of transformation is high, the expression levels of the transforming DNA is very high as well, as is illustrated by the exceptionally high resistance against G418 of the transformed Phaffia cells when the open reading frame of the G418-resistance gene was fused to a promoter according to the invention when compared to the G418 resistance gene under control of the actin promoter in pGB-Ph9. It is concluded, therefore, that the GAPDH-promoter is a high-level transcriptional promoter that can be suitably used in conjunction with any heterologous DNA sequence, in order to reach high expression levels thereof in Phaffia strains.

It will be clear that the availability of new expression tools, in the form of the recombinant DNA according to the invention, creates a wealth of possibilities for producing new and valuable biomolecules in Phaffia.

Preferably, the downstream sequence comprises an open reading frame coding for proteins of interest. For example genes already present in Phaffia, such as those involved in the carotenoid pathway, may be manipulated by cloning them under control of the high-level promoters according to the invention. Increased expression may change the accumulation of intermediates and/or end-products or change the pathway of β-carotene, cantaxanthin, astaxanthin and the like. The overexpression of the crtB gene from Erwinia uredovora will likely increase astaxanthin levels, as the product of this gene is involved in the rate limiting step. The expression of a protein of interest may also give rise to xanthophylls not known to be naturally produced in Phaffia, such as zeaxanthin. An open reading frame that may be suitably employed in such a method includes but is not limited to the one encoding the protein producing zeaxanthin (crtZ gene) obtained from Erwinia uredovora (Misawa et al.1990. J.Bacteriol. 172: 6704-6712). Other carotenoid synthesis genes can be obtained for example from Flavobacterium (a gram-positive bacterium), Synechococcus (a cyanobacterium) or Chlamydomonas or DunalielIa (algae). Obviously, carotenoid synthesis genes of a Phaffia strain, once the genes have been isolated and cloned, are suitably cloned into a recombinant DNA according to the invention and used to modify the carotenoid content of Phaffia strains. Examples of cloned carotenoid genes that can suitably be overexpressed in Phaffia, are those mentioned in FIG. 8. Particularly useful is crtE from Phycomyces blakesleanus, encoding Geranylgeranyl Diphosphate Synthase, and crtB, encoding phytoene synthase, as this step appears to be the rate-limiting step in carotenoid synthesis in Thermus thermophylus (Hoshino T. et al., 1994, Journal of Fermentation and Bioengineering 77. No. 4, 423-424). Especially preferred sources to isolate carotenoid biosynthetic genes or cDNAs from are the fungi Neurospora crassa, Blakeslea trispora. Other yeasts shown to possess cross-hybrising species of carotenoid biosynthetic genes are Cystofylobasidium, e.g. bisporidii and capitatum.

Carotenoid biosynthesis genes have also been identified in plants; these plant cDNAs or genes from plants may be used as well. Optionally, the codon usage of the Phaffia genes or cDNAs may be adapted to the preferred use in the host organism.

Of special interest according to the present invention, are the DNA sequences coding for four different enzymes in the carotenoid biosynthesis pathway of Phaffia rhodozyma, represented in the sequence listing. It will be clear to those having ordinary skill in the art, that once these DNA sequences have been made available it will be possible to bring about slight modifications to the DNA sequence without modifying the amino acid sequence. Such modifications are possible due to the degeneracy of the genetic code. Such modifications are encompassed in the present invention. However, also modifications in the coding sequences are envisaged that create modifications in the amino acid sequence of the enzyme. It is well known to those of skill in the art that minor modifications are perfectly permissible in terms of enzymatic activity. Most changes, such as delections, additions or amino acid substitutions do not affect enzymatic activity, at least not dramatically. Such variants as comprise one or more amino acid deletions, additions or substitutions can readily be tested using the complementation test disclosed in the specification. The skilled person is also familiar with the term “conservative amino acid substitutions”, meaning substitutions of amino acids by similar amino acids residing in the same group. The skilled person is also familiar with the term “allelic variant”, meaning naturally occurring variants of one particular enzyme. These conservative substitutions and allelic enzyme variants do not depart from the invention.

As stated, at the DNA level considerable variation is acceptable. Although the invention discloses four DNA sequences, as represented in SEQIDNO: 12, SEQIDNO: 14, SEQIDNO: 16, SEQIDNO: 18, SEQIDNO:20, or SEQIDNO: 22, in detail also isocoding variants of the DNA sequence represented in SEQIDNO: 12, SEQIDNO: 14, SEQIDNO: 16, SEQIDNO: 18, SEQIDNO: 20, or SEQIDNO: 22, are encompassed by the present invention. Those of skill in the art would have no difficulty in adapting the nucleic acid sequence in order to optimize codon usage in a host other than P. rhodozyma. Those of skill in the art would know how to isolate allelic variants of a DNA sequence as represented in SEQIDNO: 12, SEQIDNO: 14, SEQIDNO: 16, SEQIDNO, 11, SEQIDNO: 20, or SEQIDNO: 22 from related Phaffia strains. Such allelic variants clearly do not deviate from the present invention.

Furthermore, using the DNA sequences disclosed in the sequence listing, notably SEQIDNO: 12, SEQIDNO: 14, SEQIDNO: 16 or SEQIDNO: 18, as a probe, it will be possible to isolate corresponding genes form other strains, or other microbial species, or even more remote eukaryotic species if desired, provided that there is enough sequence homology, to detect the same using hybridisation or amplification techniques known in the art.

Typically, procedures to obtain similar DNA fragments involve the screening of bacteria or bacteriophage plaques transformed with recombinant plasmids containing DNA fragments from an organism known or expected to produce enzymes according to the invention. After in situ replication of the DNA, the DNA is released from the cells or plaques, and immobilised onto filters (generally nitro-cellulose). The filters may then be screened for complementary DNA fragments using a labeled nucleic acid probe based on any of the sequences represented in the sequence listing. Dependent on whether or not the organism to be screened for is distantly or closely related, the hybridisation and washing conditions should be adapted in order to pick up true positives and reduce the amount of false positives. A typical procedure for the hybridisation of filter-immobilised DNA is described in Chapter 5, Table 3, pp. 120 and 121 in: Nucleic acid hybridisation—a practical approach, B. D. Hames & S. J. Higgins Eds., 1985, IRL Press, Oxford). Although the optimal conditions are usually determined empirically, a few useful rules of thumb can be given for closely and less closely related sequences.

In order to identify DNA fragments very closely related to the probe, the hybridisation is performed as described in Table 3 of Hames & Higgins, supra, (the essentials of which are reproduced below) with a final washing step at high stringency in 0.1*SET buffer (20 times SET 3M NaCl, 20 mM EDTA, 0.4 M Tris-HCl, pH 7.8), 0.1% SDS at 68° Celsius).

To identify sequences with limited homology to the probe the procedure to be followed is as in Table 3 of Hames & Higgins, supra, but with reduced temperature of hybridisation and washing. A final wash at 2*SET buffer, 50° C. for example should allow the identification of sequences having about 75% homology. As is well known to the person having ordinary skill in the art, the exact relationship between homology and hybridisation conditions depend on the length of the probe, the base composition (% of G+C) and the distribution of the mismatches; a random distribution has a stronger decreasing effect on T_(m) then a non-random or clustered pattern of mismatches.

The essentials of the procedure described in Table 3, Chapter 3 of Hames & Higgins are as follows:

(1) prehybridisation of the filters in the absence of probe, (2) hybridisation at a temperature between 50 and 68° C. in between 0.1 and 4*SET buffer (depending on the stringency), 10*Denhardt's solution (100*Denhardt's solution contains 2% bovine serum albumin, 2% Ficoll, 2% polyvinylpyrrolidone), 0.1% SDS, 0.1% sodiumpyrophosphate, 50 μg/ml salmon sperm DNA (from a stock obtainable by dissolving 1 mg/ml of salmon sperm DNA, sonicated to a length of 200 to 500 bp, allowed to stand in a water bath for 20 min., and diluted with water to a final concentration of 1 mg/ml); hybridisation time is not too critical and may be anywhere between 1 and 24 hours, preferably about 16 hours (o/n); the probe is typically labeled by nick-translation using ³²P as radioactive label to a specific activity of between 5*10⁷ and 5*10⁸ c.p.m./μg; (3) (repeated) washing of the filter with 3*SET, 0.1% SDS, 0.1% sodiumpyrophosphate at 68° C. at a temperature between 50C and 68° C. (dependent on the stringency desired), repeated washing while lowering the SET concentration to 0.1%., wash once for 20 min. in 4*SET at room temperature, drying filters on 3MM paper, exposure of filters to X-ray film in a cassette at −70° C. for between 1 hour and 96 hours, and developing the film.

Generally, volumina of prehybridisation and hybridisation mixes should be kept at a minimum. All “wet” steps may be carried out in little sealed bags in a pre-heated water bath.

The above procedure serves to define the DNA fragments said to hybridise according to the invention. Obviously, numerous modifications may be made to the procedure to identify and isolate DNA fragments according to the invention. It is to be understood, that the DNA fragments so obtained fall under the terms of the claims whenever they can be detected following the above procedure, irrespective of whether they have actually been identified and/or isolated using this procedure.

Numerous protocols, which can suitably be used to identify and isolate DNA fragments according to the invention, have been described in the literature and in handbooks, including the quoted Hames & Higgins, supra).

With the advent of new DNA amplification techniques, such as direct or inverted PCR, it is also possible to clone DNA fragments in vitro once sequences of the coding region are known.

Also encompassed by the claims is a DNA sequence capable, when bound to nitrocellulose filter and after incubation under hybridising conditions and subsequent washing, of specifically hybridising to a radio-labelled DNA fragment having the sequence represented in SEQIDNO: 12, SEQIDNO: 14, SEQIDNO: 16 or SEQIDNO: 18, as detectable by autoradiography of the filter after incubation and washing, wherein said incubation under hybridising conditions and subsequent washing is performed by incubating the filter-bound DNA at a temperature of at least 50° C., preferably at least 55° C., more preferably at least 60° C. in the presence of a solution of the said radio-labeled DNA in 0.3 M NaCl, 40 mM Tris-HCl, 2 mM EDTA, 0.1% SDS, pH 7.8 for at least one hour, whereafter the filter is washed at least twice for about 20 minutes in 0.3 M NaCl, 40 mM Tris-HCl, 2 mM EDTA, 0.1% SDS, pH 7.8, at a temperature of 50° C., preferably at least 55° C., more preferably at least 60° C., prior to autoradiography.

The heterologous DNA sequence according to the invention may comprise any open reading lo frame coding for valuable proteins or their precursors, like pharmaceutical proteins such as human serum albumin, IL-3, insulin, factor VIII, tPA, EPO, α-interferon, and the like, detergent enzymes, such as proteases and lipases and the like, cell wall degrading enzymes, such as xylanases, pectinases, cellulases, glucanases, polygalacturonases, and the like, and other enzymes which may be useful as additives for food or feed (e.g. chymosin, phytases, phospholipases, and the like). Such genes may be expressed for the purpose of recovering the protein in question prior to subsequent use, but sometimes this may not be necessary as the protein may be added to a product or process in an unpurified form, for example as a culture filtrate or encapsulated inside the Phaffia cells.

The yeast cells containing the carotenoids can be used as such or in dried form as additives to animal feed. Furthermore, the yeasts can be mixed with other compounds such as proteins, carbohydrates or oils.

Valuable substances, such as proteins or pigments produced by virtue of the recombinant DNA of the invention may be extracted. Carotenoids can also be isolated for example as described by Johnson et al. (Appl. Environm. Microbiol. 35: 1155-1159 (1978)).

Purified carotenoids can be used as colorants in food and/or feed. It is also possible to apply the carotenoids in cosmetics or in pharmaceutical compositions.

The heterologous downstream sequence may also comprise an open reading frame coding for reduced sensitivity against a selective agent. The open reading frame coding for an enzyme giving G418 resistance was used satisfactorily in the method according to the invention, but the invention is not limited to this selection marker. Other useful selection markers, such as the phleomycin resistance gone may be used, as disclosed in EP 590 707. Each of these genes is advantageously expressed under the control of a strong promoter according to the invention, such as the GAPDH-promoter.

The invention is now being illustrated in greater detail by the following non-limitative examples.

EXPERIMENTAL

Strains:

E. coli DH5α: supE44lacU169 (80lacZM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1

E. coli LE392: supE44 supF58 hsdR514 galK2 galT22 metB1 trpR55 lacY1

P. rhodozyma CBS6938

Plasmids:

pUC19 (Gibco BRL)

pTZ19R

PUC-G418

pGB-Ph9 (Gist-brocades)

pMT6 (1987, Breter H.-J., Gene 53, 181-190))

Media:

LB: 10 g/l bacto tryptone, 5 g/l yeast extract, 10 g/l NaCl. Plates; +20 g/l bacto agar. When appropriate 50 μg/ml ampicillin.

YePD: 10 g/l yeast extract, 20 g/l bacto peptone, 20 g/l glucose. Plates; +20 g/l bacto agar. When appropriate 50 g/ml Geneticin (G418).

Methods

All molecular cloning techniques were essentially carried out as described by Sambrook et al. in Molecular Cloning: a Laboratory Manual, 2nd Edition (1989; Cold Spring Harbor Laboratory Press).

Enzyme incubations were performed following instructions described by the manufacturer. These incubations include restriction enzyme digestion, dephosphorylation and ligation (Gibco BRL).

Isolation of chromosomal DNA from Phaffia rhodozyma as described in example 3 of patent Gist-brocades; EP 0 590 707 A1. Chromosomal DNA from K. lactis and S.cerevisiae was isolated as described by Cryer et al.(Methods in Cell Biology 12: 39, Prescott D. M. (ed.) Academic Press, New York).

Isolation of large (>0.5-kb) DNA fragments from agarose was performed using the Geneclean II Kit whereas small (<0.5-kb) and DNA fragments or fragments from PCR mixtures were isolated using Wizard™ DNA, Clean-Up System (Promega).

Transformation of E. coli was performed according to the CaCl₂ method described by Sambrook et al. Packaging of cosmid ligations and transfection to E. coli LE392 was carried out using the Packagene Lambda DNA Packaging System (Promega), following the Promega protocols.

Isolation of plasmid DNA from E. coli was performed using the QIAGEN (Westburg B. V. NL).

Transformation of Phaffia CBS6938 was done according to the method for H. polymorpha described by Faber et al., supra;

Inoculate 30 ml of YePD with 1 CBS6938 colony

Grow 1-2 days at 21° C., 300 rpm (pre-culture)

Inoculate 200 ml of YePD with pre-culture to OD₆₀₀=between 0 and 1 (if above 1 dilute with water)

Grown o/n at 21° C., 300 rpm until OD₆₀₀=1.2 (dilute before measuring)

Centrifuge at 5 min. 8000 rpm, room temperature. Remove supernatant thoroughly

Resuspend pellet in 25 ml 50 mM KPi pH 7.0, 25 mM DTT (freshly made) Transfer suspension to a fresh sterile 30 ml centrifuge tube and incubate for 15 min. at room temperature

Centrifuge 5 min. at 8000 rpm 4° C., remove supernatant thoroughly

Resuspend pellet in 25 ml of ice cold STM (270 mM sucrose, 10 mM Tris pH 7.5, 1 mM MgCl₂)

Centrifuge 5 min. at 8000 rpm, 4° C.

Repeat washing step

Resuspend cells in 0.5 ml of ice cold STM (3*10⁹ cells/ml). Keep on ice!

Transfer 60 μl of cell suspension to pre-cooled Eppendorf tubes containing 5 μg transforming DNA (use precooled tips!), Keep on ice

Transfer Cell/DNA mix to precooled electroporation cuvettes (top to bottom)

Pulse: 1.5 kV, 400Ω, 25 μF

Immediately add 0.5 ml of ice cold YePD. Transfer back to ep using a sterile Pasteur pipette

Incubate 2.5 hrs at 21° C.

Plate 100 μl onto YePD-plates containing 40 μg/ml G418

Incubate at 21° C. until colonies appear.

Pulsed Field Electrophoresis was performed using a GENE Navigator+accessories (Pharmacia). Conditions: 0.15*TBE, 450 V, pulse time 0.5 s, 1.2% agarose, run time 2 h.

Polymerase Chain Reaction (PCR) experiments were performed in mixtures having the following composition:

5 ng of plasmid DNA or 1 μg chromosomal DNA

0.5 μg of oligo nucleotides (5 μg degenerated oligo's in combination with chromosomal DNA)

10 nm of each dNTP

2.5 m KCl

0.5 μm Tris pH 8.0

0.1 μm MgCl2

0.5 μg gelatin

1.3 U Taq polymerase (5 U in combination with chromosomal DNA)

H₂O was added to a total volume of 50 μl

Reactions were carried out in an automated thermal cycler (Perkin-Elmer). Conditions: 5 min. 95° C., followed by 25 repeated cycli; 2′ 94° C., 2′ 45° C. 3′ 72° C. Ending; 10 min. 72° C.

Fusion PCR reactions were performed as described above, except that 2 DNA fragments with compatible ends were added as a template in equimolar amounts.

Oligo nucleotide sequences were as follows:

3005: CGGGATCCAA(A/G)CTNACNGGNATGGC (SEQIDNO: 1);

3006: CGGGATCC(A/G)TAICC(C/A/G)(C/T)A(T/C)TC(A/G)TT(A/G)TC(A/G)TACCA (SEQIDNO: 2);

4206: GCGTGACTTCTGGCCAGCCACGATAGC (SEQIDNO: 3);

5126: TTCAATCCACATGATGGTAAGAGTGTTAGAGA (SEQIDNO: 4);

5127: CTTACCATCATGTGGATTGAACAAGATGGAT (SEQIDNO: 5);

AAGCTCTCGAGGTACCTGGTGGGTGCATGTATGTAC (SEQIDNO: 6);

CCAAGGCCTAAAACGGATCCCTCCAAACCC (SEQIDNO: 7);

GCCAAGCTTCTCGAGCTTGATCAGATAAAGATAGAGAT (SEQIDNO: 8);

EXAMPLE 1 G-418 Resistance of Phaffia Transformant G418-1

To determine the expression of the G418 resistance gene in pGB-Ph9, transformant G418-1 (EP0 590 707 A1) was exposed to increasing concentrations of G418. Two dilutions of a G418-1 culture were plated onto YepD agar containing 0-1000 jig/ml G418 (Table 1).

TABLE 1 Survival of Phaffia transformant G418-1 on YepD agar medium containing increasing concentrations of G418. Phaffia G418-1 Phaffia G418-1 Phaffia (CBS6938) Dil. = 10⁻⁴ Dil. = 10⁻⁵ Dil. = 0 [G418] μg/ml (OD₆₀₀ = 7) (OD₆₀₀ = 7) (OD₆₀₀ = 5) 0 >300 74 >300 200 >300 70 0 300 >300 61 0 400 212 13 0 500 10 2 0 600 0 0 0 700 0 0 0 800 0 0 0 900 0 0 0 1000 0 0 0

At a concentration of 600 μg/ml G418 less than 1% of the plated cells survived. It can be concluded that despite multicopy integration of pGB-Ph9, G418-1 shows a rather weak resistance to G418 (Scorer et al, 1994, Bio/Technology 12, p. 181 et seq., Jimenez and Davies, 1980, Nature 187 p. 869 et seq.), most probably due to a weak action of the Phaffia actin promoter in the plasmid. The results that the Phaffia actin promoter works poorly, prompted us to isolate promoter sequences of Phaffia with strong promoter activity.

EXAMPLE 2

Synthesis of Specific Probes of Glycolytic Genes from Phaffia rhodozyma by PCR

The polymerase chain reaction (PCR) technique was used in an attempt to synthesize a homologous probe of the genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK) and the triose phosphate isomerase (TPI) of Phaffia rhodozyma.

A set of degenerated oligonucleotides was designed based on the conserved regions in the GAPDH-gene (Michels et al., 1986. EMBO J. 5: 1049-1056), PGK-gene (Osinga et al., 1985. EMBO J. 4: 3811-3817) and the TPI-gene (Swinkels et al., 1986. EMBO J. 5: 1291-1298).

All possible oligo combinations were used to synthesize a PCR-fragment with chromosomal DNA of Phaffia rhodozyma (strain CBS6938) as template. Chromosomal DNA of Saccharomyces cerevisiae and Kluyveromyces lactis as template was used to monitor the specificity of the amplification. The PCR was perfomed as described above, the PCR conditions were 1′ 95° C., 2′ annealing temperature (T_(a)), in 5′ from annealing temperature to 72° C., 2′ 72° C., for 5 cycli followed by 1′ 95° C., 2′ 55° C. and 2′ 72° C. for 25 cycli and another elongation step for 10′ 72° C. Three different T_(a) were used 40° C., 45° C. and 50° C.

Under these conditions, only one primer combination produced a fragment of the expected size on chromosomal DNA of Phaffia as template. Using the oligo combination no: 3005 and 3006 and a T_(a) of 45° C. a 0.3-kb fragment was found. Specifically, the GAPDH oligonucleotides correspond with amino acids 241-246 and 331-338 of the published S. cerevisiae sequence. (It was concluded that to isolate the promoters corresponding to the PGK- and TPI-genes from Phaffia, further optimization of the PCR-conditions is required, or homologous primers should be used. Another alternative method for isolating high level promoters is disclosed in the detailed description, supra.

The amplified fragment was purified from the PCR reaction and was digested with BamHI and ligated into the dephosphorylated BamHI site of pTZ19R. The ligation mixture was transformed to competent E. coli DH5α cells prepared by the CaCl₂-method and the cell were plated on LB-plates with 50 μg/ml Amp and 0.1 mM IPTG/50 μg/ml X-gal. Plasmid DNA was isolated from the white colonies. The pTZ19R clone with the right insert, called pPRGDH1, was subsequently used for sequence analysis of the insert. The cloned sequence encoded for the carboxy terminal fragment of GAPDH of Phaffia as shown by comparison with the GAPDH-gene sequence of S. cerevisiae (Holland and Holland, 1979. J. of Biol. Chem. 254: 9839-9845).

EXAMPLE 3 Isolation of the GAPDH-gene of Phaffia

To obtain the complete GAPDH-gene including expression signals the 0.3-kb BamHI fragment of pPRGDH1 was used to screen a cosmid library of Phaffia.

Preparation of the Vector for Cosmid Cloning

Vector preparation was simplified, because of the presence of a double cos-site in pMT6. pMT6 was digested to completion with blunt end cutter PvuII to release the cos-sites. Digestion efficiency was checked by transformation to E. coli DH5α and found to be >99%.

The PvuII digested pMT6 was purified by phenol:chloroform extraction and ethanol precipitation and finally solved in 30 μl TE at a concentration of 2 μg/μl. The vector was subsequently digested with cloning enzyme BamHI and the vector arms were purified as described above (“Experimental”).

Preparation of Target DNA

Isolation of genomic DNA of Phaffia strain CBS6938 was performed as described in the part named “Experimental”. The cosmid pMT6 containing inserts of 25-38-kb are most efficiently packaged. Therefore genomic DNA was subjected to partial digestion with the restriction enzyme Sau3A. Target DNA was incubated with different amounts of enzyme. Immediately after digestion the reactions were stopped by the extraction of DNA from the restriction mixture with phenol-chloroform. The DNA was Is precipitated by using the ethanol method and the pelleted DNA after centrifugation was dissolved in a small volume of TE. Contour clamped homogeneous electric field (CHEF) electrophoresis was used to estimate the concentration and size of the fragments (Dawkins, 1989, J. of Chromatography 492, pp.

Construction of Genomic Cosmid Library

Ligation of approximately 0.5 μg of vector arm DNA and 0.5 μg of target DNA was performed in a total volume of 10 μl in the presence of 5 mM ATP (to prevent blunt end ligation). Packaging in phage heads and transfection to E. coli LE 392 as described in Experimental. The primary library consisted of 7582 transfectants with an average insert of 28-kb as determined by restriction analysis. The library represents 3.5 times the genome with a probability of the presence of all genes in the library of 0.97 as calculated according to Sambrook (supra). For library amplification the transfectants were pooled by resuspending in 8 ml LB-broth. Additional 4.8 ml glycerol was added. The transfectants mixture was divided into 16 samples of 800 μl each and stored at −80° C. This amplified library consisted of 2.9*10⁹ transfectants.

Screening of the Cosmid Library

A 100 μl sample was taken from this library and further diluted (106) in LB-broth and 200 μl was plated onto 10 LB-plates containing ampicillin. The plates were incubated overnight at 37° C. Each plate contained 300-400 colonies and filters were prepared. These filters were screened with the GAPDH-probe using hybridization and washing conditions as described above (“Experimental”). After 16 hours exposure, 3 strong hybridization signals were found on the autoradiogram. Cosmid DNA isolated from these positive colonies was called pPRGDHcos1, pPRGDHcos2 and pPRGDH cos3.

Chromosomal DNA isolated from Phaffia rhodozyma strain CBS 6938 and cosmid pPRGDHcos1 was digested with several restriction enzymes. The DNA fragments were separated, blotted and hybridized as described before. The autoradiograph was exposed for different time periods at −80° C. The film showed DNA fragments of different length digested by different restriction enzymes which hybridize with the GAPDH-probe (FIG. 1).

Furthermore, from Southern analysis of the genomic DNA of Phaffia using the GAPDH fragment as probe, it was concluded that the GAPDH-encoding gene is present as a single copy gene in Phaffia rhodozyma, whereas in Saccaromyces cerevisiae GAPDH is encoded by three closely related but unlinked genes (Boucherie et al., 1995. FEMS Microb. Letters 135:127-134).

Hybridizing fragments of pPRGDHcos1 for which a fragment of the same length in the chromosomal DNA digested with the same enzyme was found, were isolated from an agarose gel. The fragments were ligated into the corresponding sites in pUC19. The ligation mixtures were transformed to competent E. coli cells. The plasmids with a 3.3-kb Sa/I insert and a 5.5-kb EcoRI insert were called pPRGDH3 and pPRGDH6, respectively. The restriction map of pPRGDH3 and pPRGDH6 is shown in FIG. 2. Analysis of the sequence data of the insert in pPRGDH1 showed us that there was a HindIII site at the C-terminal part of the GAPDH-gene. From this data it was suggested that the insert in pPRGDH6 should contain the complete coding sequence of GAPDH including promoter and terminator sequences.

EXAMPLE 4 Characterization of the GAPDH-gene

In order to carry out sequence analysis without the need to synthesize a number of specific sequence primers a number of deletion constructs of plasmids pPRGDH3 and pPRGDH6 were made using convenient restriction sites in or near the putative coding region of GAPDH gene.

The plasmids were digested and after incubation a sample of the restriction mixture was analyzed by gel electrophoresis to monitor complete digestion. After extraction with phenol-chloroform the DNA was precipitated by ethanol. After incubation at −20° C. for 30′ the DNA is pelleted by centrifugation, dried and dissolved in a large volume (0.1 ng/μl) of TE. After ligation the mixtures were transformed to E. coli. Plasmid DNA isolated from these transformants was analyzed by restriction analysis to reveal the right constructs. In this way the deletion constructs pPRGDH3δHIII, pPRGDH6δBamHI, pPRGDH6δSstI and pPRGDH6δSalI (FIG. 1).

In addition to this, the 0.6-kb and 0.8-kb SstI fragments derived from pPRGDH6 were subcloned in the corresponding site of pUC 19. Sequence analysis was carried out using pUC/M3 forward and reverse primers (Promega). The sequencing stategy is shown in FIG. 2 (see arrows).

On the basis of homology with the GAPDH-gene sequence of S. cerevisiae (Holland and Holland, 1979. J. of Biol. Chem. 254: 9839-9845) and K. lactis (Shuster, 1990. Nucl. Acids Res. 18, 4271) and the known splice site concensus J. L. Woolford. 1989. Yeast 5: 439-457), the introns and the possible ATG start were postulated.

The GAPDH gene has 6 introns (FIG. 1) and encodes a polypeptide of 339 amino acids. This was completely unexpected considering the genomic organisation of the GAPDH genes of K. lactis and S. cerevisiae which have no introns and both consist of 332 amino acids. The homology on the amino acid level between the GAPDH gene of Phaffia and K. lactis and S. cerevisiae is 63% and 61%, respectively. Most of the introns in the GAPDH gene are situated at the 5′ part of the gene. Except intron III all introns contain a conserved branch-site sequence 5′-CTPuAPy-3′ found for S. cerevisiae and S. pombe.

By computer analysis of the upstream sequence using PC-gene 2 putative eukaryotic promoter elements, TATA-box (position 249-263 in SEQIDNO: 11) and a number of putative Cap signal (between position 287 and 302 in SEQIDNO: 11) were identified.

EXAMPLE 5 Cloning of the GAPDH Promoter Fused to G418 in pUCG418

In order to construct a transcription fusion between the GAPDH promoter and the gene encoding G418 resistence the fusion PCR technique was used. Using plasmid pPRGDH6 the GAPDH promoter could be amplified by standard PCR protocols (“Experimental”).

In the PCR mix pPRGDH6 and oligo's No. 5177 and 5126 (Sequences in “Experimental”) were used. A 416 bp DNA fragment was generated containing the entire GAPDH promoter sequence. In addition this fragment also contains a HindIII, XhoI and a KpnI restriction site at it's 5′end and 12 nt overlap with the 5′ end of the gene encoding G418 resistance.

The 217 bp portion of the 5′ end of the G418 coding sequence was also amplified by PCR using pUC-G418 and oligo's 4206 and 5127. A 226 bp DNA fragment was obtained containing the 217 bp 5′ end of G418 and having a 9 nucleotides overlap with the 3′ end of the earlier generated GAPDH promoter fragment. It also contained a MscI site at it's 3end.

The PCR fragments were purified from the PCR mixture using the WIZARD Kit. Approximately 1 μg of the GAPDH promoter fragment and 1 μg of the G418 PCR fragment were used together with oligo's 5177 and 4206 in a fusion PCR experiment (Experimental). A 621 bp DNA fragment was generated, containing the GAPDH promoter directly fused to the 5′ portion of G418. After purification the DNA fragment was digested with MscI and KpnI. The 3.4 Kb MscI-KpnI fragment of pUC-G418, containing pUC sequences and the 3′ portion of G418, was used as a vector. The ligation mixture was transformed to competent E. coli DH5 α cells. Transformant colonies containing the fusion PCR DNA inserted were identified by digestion with different restriction enzymes.

Thus, placid pPR1 was obtained containing the GAPDH promoter directly fused to the G418 marker gene. Three pPR1 vectors isolated from independent transformants were used in further cloning experiments.

To target the plasmid, after transformation, to a specific integration site a 3.0-kb SstI1 fragment containing a part of the ribosomal DNA of Phaffia was cloned in pPR1. The ribosomal DNA fragment was isolated from an agarose gel after digestion with Sst1 of plasmid pGB-Ph11 (EP 590 707 A1). This fragment was ligated in the dephosphorylated Sst1 site of pPR1. The ligation mixture was transformed to competent E. coli cells. Plasmid DNA was isolated and using restriction analysis it was shown that several colonies contain the expected plasmid pPR2. The complete cloning strategy is shown in FIG. 3.

EXAMPLE 6 Transformation of Phaffia with pPR2

Transformation of Phaffia strain 6938 was performed using an electroporation procedure as previously described by Faber et al. (1994, Curr. Genet. 1994: 2,305-310) with the following modifications:

Electropulsing was performed using the Bio-rad Gene Pulser with Pulse Controller and with Bio-rad 2 mm cuvettes.

Phaffia was cultivated for 16 h at 21° C.

Per transformation 2×10⁸ cells were used together with 5 μg of linearized vector. Linearization was done in the rDNA sequence using ClaI to enable integration at the rDNA locus in the Phaffia genome. Following the electric pulse (7.5 kV/cm, 400Ω and 25 μF) 0.5 ml YePD medium was added to the cell/DNA mixture. The mixture was incubated for 2.5 h at 21° C. and subsequently spread on 5 selective YEDP agar plates containing 40 μg/ml G(418.

As shown in Table 2 we were able to generate transformants with 115 transformants per μg DNA; the average transformation frequency was 50 transformants/g pPR2 as judged over a number of experiments. Transformation of the closed circular of pPR2 did not result in transformation suggesting that there is no autonomously replicating sequence present within the vector sequences. Using pPR2 a 10 to 50-fold increase in transformation frequency was found compared to a previous constructed transformation vector for Phaffia, called pGB-Ph9. In this latter vector a translation fusion was made between the 5′ part of the actin gene of Phaffia and G418.

In order to analyze the level of resistance of transformants the mixture or DNA/cells was plated onto selective plates containing different amounts of G418. Although the total number of transformants decreases with the increasing amounts of G418, we were still able to obtain a considerable number of transformants (table 3).

In another experiment 30 transformants obtained under standard selection conditions (40 μl/ml) were transferred to plates containing 50, 200 or 1000 μg/ml . After incubation of the plates at 21° C. for 4-5 days, 23 transformants out of 30 tested were able to grow on plates containing 200 μg/ml G418. One transformant was able to grow on plates containing upto and above 1000 μg/ml G418.

TABLE 2 Transformation frequency of pGB-Ph9 and pPR2. Exp. 1 Exp. 2 —  69  8 pGB-Ph9xBg/II  46  7 pPR2 ccc n.d n.d pPR2(A)xClaI 714  56 (B) 639 124

Total number of transformants (<1 mm) in different transformation experiments after 4-5 days incubation.

TABLE 3 Comparison of G418 sensitivity as a result of two different G418-resistance genes in pGB-Ph9 and pPR2 Number of concentration transformants G418 (μg/ml) pPR2xClaI PGB-Ph9xBg/II (=pYac4) 40 480 2 50 346 — 60 155 — 70 61 — 80 141 — 90 72 — 100 64 —

Analysis of pPR2 Transformants

To analyse the integration event and the number of integrated vector copies total genomic DNA from six independent transformants was isolated. Therefore these transformants were cultivated under selective conditions, i.e. YePD+50 μg/ml G418. Chromosomal DNA was digested with ClaI. The DNA framgments were separated by gel electrophoresis and transfered to nitrocellulose and the Southern blot was probed with Phaffia DNA.

Besides the rDNA band of 9.1 kb an additional band of 7.1 kb of similar fluorescing intensity was observed in the transformants. This band corresponds to the linearised form of pPR2. From the intensity of these bands it was concluded that the copy number was about 100-140 copies of pPR2. These results are similar to those observed for pGB-Ph9, ruling out that the improved G418-resistance is due to differences in copy number of integrated vectors alone. It is not known whether the multiple copy event is caused by multiple copy integration of pPR2 or by the amplification of a single copy in the rDNA or a combination of both events.

EXAMPLE 7 Construction of pPR2T by Cloning the GAPDH-terminator into pPR2

Eukaryotic mRNAs contain modified terminal sequences, specificaly the 3′ terminal poly(A). As the prokaryotic gene encoding G418 resistance lacers eukaryotic termination signals, which might effect proper transcription termination and mRNA stability (1994, Raue, H. A., TIBTECH 12: 444-449), a part of the 3′ non-coding sequence of GAPDH was introduced. To that end, a 307 bp fragment, consisting of 281 bp of the 3′ non-coding region of GAPDH and other additional cloning sequences, was amplified by PCR using the oligo's 5137 and 5138 (“Experimental”). The upstream oligo 5137 consists of the last 14 nucleotide& of the coding and 17 nucleotides of the 3′ non-coding region of GAPDH. By base substitutions of the 5th (T→A) and 8th (T→C) nucleotide of the non-coding sequence a BamHI restriction site was introduced. In addition this fragment contains a XhoI and a HindIII restriction site at its 3′ end.

The PCR fragment was purified from the PCR mixture using the WIZARD Purification Kit and digested with BamHI and HindIII. A 288 bp fragment was isolated and cloned into the corresponding sites of the previously constructed Phaffia transformation vector pPR2, yielding pPR2T.

Upon transformation of Phaffia, using G418 as selective agent, the transformation frequencies (number of transformants per jig of DNA) obtained with the improved construct pPR2T was approximately 5 to 10 times higher than the transformation frequency of pPR2 (i.e. without a Phaffia homologous transcription termination signal). The results of a typical experiment are given in Table 4.

TABLE 4 Transformation frequency at 50 μg/ml G418 for PGB-Ph9 pPR2 and pPR2T Vector transformants transformants/μg DNA pGB-Ph9 (ccc) — — pGB-Ph9 (xBg/II) 60 1 pPR2 (ccc) 1 — pPR2 (xClaI) 3000-9600 50-160 pPR2T (ccc) — — pPR2T (xClaI) 45600 760 pPR2T (xSfil) 1080 18

Phaffia cells transformed with pPR2T were tested for their ability to grow on high levels of G418. The level of G418 on which growth is still possible was taken as a measure of the expression level of the G418 resistance gene in transformants, as a result of the presence of the Phaffia promoter, and/or terminator. Preliminary results indicate that the number of transformants able to grow on high levels of G418 are significantly higher than without terminator.

In Summary

From the above results, it was concluded, that the presence of the GAPDH-promoter (pPR2) resulted in a considerable increase of the transformation frequency (from 1 to at least 50 per μg of DNA) when compared to the vector containing the actin-promoter (pGB-Ph9). These results are in line with the results obtained with the G418 sensitivity test (Table 3 and 4) which indicate superior expression levels under the control of the GAPDH promoter. The possibility that the difference in transformation frequency could be due solely to the difference in linearising the vectors, (BglII, ClaI and SfiI all cut inside the ribosomal DNA locus, but at different positions), was ruled out by comparison of pPR2(xSfiI) with pGB-Ph9(xSfiI). The difference in transformation frequency between the two pPR2 and pGB-Ph9, linearised with SfiI is still considerable. However, it is concluded that the choice of the linearisation site does have effect on the transformation frequency; linearisation with ClaI is preferred.

The improvements obtained by using a high-level promoter, such as GAPDH, are irrespective of whether a homologous terminator is used (pPR2 (without homologous terminator) performs far better than pGB-Ph9, both in G418 sensitivity tests, as well as in terms of transformation frequency).

The presence of a homologous terminator results in both higher transformation frequencies and higher expression levels; this result is concluded to be independent of the promoter used. Preliminary results indicate that considerable improvements are obtained when the pGB-Ph9 construct is completed with a transcription terminator, such as the GAPDH-terminator used in pPR2T.

The following Examples illustrate the isolation of DNA encoding enzymes involved in the carotenoid biosynthesis pathway of Phaffia rhodozyma. These DNA sequences can suitably be used for a variety of purposes; for example to detect and isolate DNA sequences encoding similar enzymes in other organisms, such as yeast by routine hybridisation procedures, to isolate the transcription promoters and/or terminators, which can be used to construct expression vectors for both heterologous as well as homologous downstream sequences to be expressed. The DNA sequences encoding carotenoid biosynthesis genes can suitably be used to study the over-expression, either under the control of their own promoters or heterologous promoters, such as the glycolytic pathway promoters illustrated above. For example transformation of Phaffia rhodozyma with carotenoid encoding DNA sequences according to the invention effectively results in amplification of the gene with respect to the wild-type situation, and as a consequence, thereof to overexpression of the encoded enzyme. Hence, the effect of over-expression of one or more genes encoding carotenoid biosynthesis genes can thus be studied. It is envisaged that mutant Phaffia strains can be obtained producing higher amounts of valuable carotenoids, such as B-carotene, cantaxanthin, zeaxanthin and/or astaxanthin. Similarly, the DNA sequences encoding enzymes involved in the carotenoid biosynthesis pathway can be introduced into other hosts, such as bacteria, for example E. coli, yeasts, for example species of Saccharomyces, Kluyveromyces, Rhodosporidium, Candida, Yarrowia, Phycomyces, Hansenula, Picchia, fungi, such as Aspergillus, Fusarium, and plants such as carrot, tomato, and the like. The procedures of transformation and expression requirements are well known to persons skilled in these arts.

Strains:

E. coli XL-Blue-MRF′Δ(mcrA)183α(mcrCB-hsdSMR-mrr) 173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac[F′ proAB laq^(q)ZΔM15 Tn10 (Tet^(r))]

ExAssist™ interference-resistant helper phage (Stategene®)

P. rhodozyma CBS6938 or

P. rhodozyma asta 1043-3

Plasmids used for Cloning:

pUC19 Ap^(r) (Gibco BRL)

Uni-ZAP™ XR vector (lambda ZAP®II vector digested with EcoRI-XhoI, CIAP treated;Strategene®)

Media:

LB: 10 g/l bacto tryptone, 5 g/l yeast extract, 10 g/l NaCl. Plates; +20 g/l bacto agar.

When appropriate 50-100 μg/ml ampicillin (Ap), 30 μg/ml chloramphenicol (Cm) and 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) was added.

YePD: 10 g/l yeast extract, 20 g/l bacto peptone, 20 g/I glucose. Plates: +20 g/l bacto agar.

All molecular cloning techniques were essentially carried out as described by Sambrook et al. in Molecular Cloning: a Laboratory Manual, 2nd Edition (1989; Cold Spring Harbor Laboratory Press). Transformation of E. coli was performed according to the CaCl₂ method described by Sambrook et al.

Enzyme incubations were performed following instructions described by the manufacturer. These incubations include restriction enzyme digestion, dephosphorylation and ligation (Gibco BRL). Isolation of plasmid DNA from E. coli was performed using-the QIAGEN (Westburg B. V. NL).

For sequence analysis deletions constructs and oligonucleotides were made to sequence the complete sequence using a Taq DYE Primer Cycle Sequencing kit (Applied Biosystems).

EXAMPLE 8 Description of Plasmids

Plasmids (pACCAR25ΔcrtE, pACCAR25ΔcrtB, pACCRT-EIB, pACCAR16ΔcrtX and pACCAR25ΔcrtX), which contain different combinations of genes involved in the biosynthesis of carotenoid in Erwinia uredovora were gifts from Prof. Misawa; Kirin Brewery co., LTD.; Japan). The biosynthetic route of carotenoid synthesis in Erwinia uredovora is shown in FIG. 8.

In addition a derivative of pACCAR25ΔcrtX, designated pACCAR25ΔcrtXΔcrtX, was made in our laboratory. By the introduction of a frameshift in the BamHI restriction Site the ertl gene was inactivated. E. coli strains harboring this plasmid acummulate phytoene which can be monitored by the red phenotype of the colony.

All plasmids are derivatives of plasmid pACYC184 (Rose RE; Nucl. Acids Res. 16 (1992) 355), which contains a marker conferring chloramphenicol-resistance. Furthermore these plasmids and derivatives thereof contain a replication origin that is compatible to vectors such as pUC and pBluescript. Each plasmid contain a get of carotenoid biosynthetic genes of Erwinia uredovora mediating the formation of different carotenoid in E. coli. The complete list of plasmid used in this study is shown in Table 5.

TABLE 5 Summary of carotenoid producing E. coli strains used in this study. CAROTENOID COLOR PLASMID: GENOTYPE: ACCUMULATED: PHENOTYPE: pACCAR25ΔcrtE crtB; crtI; crtY; farnesyl white crtX; pyrophosphate/iso- crtZ pentenyl pyrophosphate pACCAR25ΔcrtB crtE; crtI; crtY; geranylgeranyl white crtX; pyrophosphate crtZ pACCAR25ΔcrtX crtE; crtB; phytoene white ΔcrtI crtY; crtZ pACCRT-EIB crtE; crtB; crtI lycopene red pACCAR16ΔcrtX crtE; crtB; crtI β-carotene yellow crtY pACCAR25ΔcrtX crtE; crtB; crtI; zeaxanthin yellow/ crtY; orange crtZ Genes encoding: crtE, geranylgeranyl pyrophosphate synthase; crtB, Phytoene synthase; crtI, phytoene desaturase; crtY, lycopene cyclase; crtX, β-carotene hydroxylase; crtZ, zeaxanthin glycosylase

EXAMPLE 9 Construction of cDNA Library of Phaffia rhodozyma

a) Isolation of Total RNA from Phaffia rhodozyma

All solutions were made in DEPC-treated distilled water and all equipments were soaked overnight in 0.1% DEPC and then autoclaved.

A 300 ml Erlemeyer containing 60 ml YePD culture medium was inoculated with Phaffia rhodozyma strain CBS6938/1043-3 from a preculture to a final OD₆₀₀ of 0.1. This culture was incubated at 21° C. (300 rpm) until the OD₆₀₀ had reached 3-4.

The cells were harvest by centrifugation (4° C., 8000 rpm, 5 min) and were resuspended in 12 ml of ice-cold extraction-buffer (0.1 M Tris-HCl, pH 7.5; 0.1 M LiCl; 0.1 mM EDTA). After centrifugation cells were resuspended in 2 ml of ice-cold extraction-buffer, 4 g of glassbeads (0.25 mm) and 2 ml phenol were added.

The mixture was vortexed 5 times at maximum speed for 30 s with 30 s cooling incubation intervals on ice.

The cell/glassbeads/phenol mixture was centrifuged (5 min, 15.300 rpm, 4° C.) and tho aqueous phase (sup 1) was transferred to a fresh tube and was kept on ice.

The phenolic phase was retracted by adding an additional volume of 1 ml extraction buffer and 2 ml phenol.

After centrifugation (5 min, 15.300 rpm, 4° C.). the aqueous phase was transferred to sup 1 and extracted with an equal volume phenol:chloroform.

After centrifugation (5 min, 15.300 rpm, 4° C.), the aquaous phase was transferred to a fresh tube and 0.1 volume of 3 M NaAc; pH5.5 and 2. 5 volumes of EtOH was added to precipitate RNA (incubation overnight −20° C.).

The precipitate was collected by centrifugation (10 min. 15.300 rpm, 4° C.) and drained off excess liquid and the RNA pellet was washed with 70% icecold EtOH.

After removing excess liquid the RNA was resuspended in 200-800 μl DEPC-treated water. RNA was stored at −70° C. A 60 ml culture yielded 400-1500 μg total RNA. The integrity of total RNA was checked by formaldehyde RNA gel electrophoresis.

b) Selection of poly(A)⁺RNA

Isolation of poly(A)⁺ from total RNA was carried out essential as described by Sambrook et al., 1989 (Molecular cloning, a laboratory manual, second edition) using the following solutions. All solutions were prepared in DEPC-treated water and autoclaved.

RNA denaturation buffer; 1 M NaCl; 18% (v/v) DMSO.

Column-loading buffer (HEND): 10 mM Hepes, pH 7.6; 1 mM EDTA; 0.5 M Na Cl; 9% (v/v) DMSO.

Elution buffer (HE): 10 mM Hepes, pH 7.6; 1 mM EDTA.

Oligo(dT)-cellulose Type 7 was supplied by Pharmacia Biotech. 0.1 g (dry weight) of oligo(dT)-cellulose was add to 1 ml HEND and the suspension was gently shaked for 1 h at 4° C. Total RNA (1.5 mg dissolved in 500 μl) and 1 ml 1 M NaCl; 18%/ (v/v) DMSO was heated to 65° C. for 5 min. Then 600 μl NaCl/DMSO was added to the RNA, mixed and placed on ice for 5 min. The poly(A)⁺ isolation was carried out be two cycles of purification. The final yield was about 45 μg poly(A)⁺ RNA.

c) cDNA Synthesis

cDNAs were synthesized from 7.5 μg poly(A)⁺-RNAs using the cDNA Synthesis Kit (#200401;

Strategene®). Synthesis was carried out according to the instruction manual with some minor modification.

SuperScript™ II RNase H⁻ Reverse Transcriptase (Gibco BRL) was used in the first strand reaction instead of MMLV-RT.

The following reagents were add in a microcentrifuge:

3 μl of poly(A)⁺ RNAs

2 μl of linker-primer

23.5 μl DMQ

Incubate 10 min 70° C., spin quickly in microcentrifuge and add,

10 μl of 5×First Strand Buffer (provided by Gibco BRL)

5 μl of 0.1 M DTT (provided by Gibco BRL)

3 μl of first strand methyl nucleotide mixture

1 μl of RNase Block Ribonuclease Inhibitor (40 U/μl)

Annealling of template and primers by incubation the mixture at 25° C. for 10 min followed by 2 min at 42° C. and finally add;

2.5 μl SuperScript™ II RNase H⁻ Reverse Transcriptase

First-strand reaction was carried out at 42° C. for 1 h.

Size fractionation was carried out using Geneclean® II kit (supplied BIO 101, Inc.). The volume of the cDNA mixture obtained after XhoI digestion was brought up by adding DMQ to a final volume of 200 μl. Three volumes of NaI was added and the microcentrifuge tube was placed on ice for 5 min. The pellet of glassmilk was washed three times using 500 l New Wash. Finally the cDNA was eluted in 20 μl DMQ.

The yield of cDNA was about 1 μg using these conditions.

d) cDNA Cloning

cDNA library was constructed in the Uni-ZAP™ XR vector using 100 ng cDNAs. Ligation was performed two times overnight incubation at 12° C. The cDNA library was packaged using the Packagene® lambda DNA packaging system (Promega) according to the instruction manual. The calculated titer of the cDNA library was 3.5 10⁶ pfu.

e) Mass excission

Mass excision was carried out described in the protocol using derivatives of E. coli XL-Blue-MRF′ as acceptor strain (see Table 5). Dilution of cell mixtures were plated onto 145 mm LB agar plates containing ampicillin, chloramphenicol and IPTG, yielding 250-7000 colonies on each plate. The plates were incubatied overnight at 37° C. and further incubated one or two more days at room temperature.

EXAMPLE 10 Cloning of the Geranylgeranyl Pyrophosphate Synthase Gene (crtE) of Phaffia rhodozyma

a) Isolation of cDNA Clone

The entire library was excised into a farnesylpyrophosphate/isopentenyl pyrophosphate accumulating cells of E. coli XL-Blue-MRF, which carries the plasmid pACCAR25ΔcrtE (further indicated as XL-Blue-MRF′[pACCAR25ΔcrtE]). The screening for the crtE gene was based on the color of the transformants. Introduction of the crtB gene in a genetic background of XL-Blue-MRF′[pACCAR25ΔcrtE] would result in a restoration of the complete route for the biosynthesis of zeaxanthin-diglucoside, which could be monitored by the presence of a yellow/orange pigmented colony. About 8.000 colonies were spread on LB agar plates containing appropriate antibiotics and IPTG. One colonie was found to have changed to a yellow/orange color.

b) Characterization of Complementing cDNA Clone

These colonies were streaked on LB-ampicillin agar plates. Plasmid DNA was isolated from this yellow colonies and found to include a 1.85 kb fragment (FIG. 2A). The resulting plasmid, designated pPRcrtE, was used for retransformation experiments (Table 6). Only the transformation of XL-Blue-MRF′[pACCAR25ΔcrtE] with pPRcrtE resulted in a white to yellow color change in phenotype. To test whether the color change was due to complemention and not caused by cDNA alone pPRcrtE was transformed into XL-Blue-MRF′. Selection of transformants on LB-ampicillin agar plate containing IPTG did not result in color changes of the colonies (Table 6). Therefore we tentatively concluded, that we have cloned a cDNA of P. rhodozyma encoding GPPP synthase which is involved in the conversion of IPP and FPP to GGPP.

TABLE 6 Color phenotype of carotenoid producing E. coli strains transformed with pPRcrtE. pUCI9 (control) pPRcrtE XL-Blue-MRF′ white white (Ap, IPTG) XL-Blue-MRF′ white yellow/orange [pACCAR25ΔcrtE] (Ap, Cm, IPTG) XL-Blue-MRF′ white white [pACCAR25ΔcrtB] (Ap, Cm, IPTG)

Transformation: 10 ng of each plasmid was mixed to CaCl, competent E. coli cells. Transforment cells were selected by plating 1/10 and 1/100 volume of the DNA/cell mixture on LB agar-medium containing the appropriate antibiotics (in brackets).

c) Sequence Analysis of cDNA Fragment

Plasmid pPRcrtE was used to determine the nucleotide sequence of the 1.85 kb cDNA.

The sequence comprised 1830 nucleotides and a 31 bp poly(A) tail. An open reading frame (ORF) of 375 amino acids was predicted. The nucleotide sequence and deduced amino acid sequence are shown as SEQIDNO NO: 14 and 15, respectively. A search in SWISS-PROT protein sequence data bases using the Blitz amino acid sequence alignment program indicated amino acid homology (52% in 132 aa overlap; Neurospora crassa) especially to the conserved domain I in geranylgeranyl-PPi synthase enzymes of different organisms (Botella et al., Eur. J. Biochem. (1995) 233; 238-248).

EXAMPLE 11 Cloning of the Phytoene Synthase Gene (crtB) of Phaffia rhodozyma

Isolation of cDNA Clone

The entire library was excised into a geranylgeranylpyrophosphate accumulating cells of E. coli XL-Blue-MRF′, which carries the plasmid pACCAR25ΔcrtB (further indicated as XL-Blue-MRF′[pACCAR25ΔcrtB]). The screening for the crtB gene was based on the color of the transformants.

Introduction of the crtB gene in a genetic background of XL-Blue-MRF′[pACCAR25ΔcrtB] would result in a restoration of the complete route for the biosynthesis of zeaxanthin-diglucoside, which could be monitored by the presence of a yellow/orange pigmented colony.

About 25.000 colonies were incubated on LB agar plates containing appropriate antibiotics and IPTG. Three colonies were found to have changed to a yellow/orange color.

b) Characterization of Complementing cDNA Clone

These colonies were streaked on LB-ampicillin agar plates. Plasmid DNA, designated pPRcrtB1 to 3, was isolated from these yellow colonies and found to include a 2.5 kb fragment (FIG. 2B). One of the resulting plasmids, pPRcrtB1 was used for retransformation experiments (Table 7). Only the transformation of XL-Blue-MRF′[pACCAR25ΔcrtB] with pPRcrtB resulted in a white to yellow color change in phenotype. Therefore we tentative conclude that we have cloned a cDNA of P. rhodozyma encoding phytoene synthase which is involved in the conversion of 2 GGPP molecules via prephytoene pyrophosphate into phytoene.

TABLE 7 Color phenotype of carotenoid producing E. coli strains transformed with pPRcrtB. pUC19 (control) pPRcrtB XL-Blue-MRF′ white white (Ap, IPTG) XL-Blue-MRF′ white yellow/orange [pACCA25ΔcrtB (Ap, Cm, IPTG) XL-Blue-MRF′ white white [pACCA25ΔcrtE (Ap, Cm, IPTG)

Legend: see Table 6.

c) Sequence Analysis of cDNA Fragment

Plasmid pPRcrtB2, which contains the longest cDNA insert, was used to determine the nucleotide sequence of the 2.5 kb cDNA. The sequence comprised 2483 nucleotides and a 20 bp poly(A) tail. An open reading frame (ORF) of 684 amino acids was predicted. The nucleotide sequence and deduced amino acid sequence are shown in SEQIDNOs: 12 and 11, respectively. A search in SWISS-PROT protein sequence data bases using the Blitz amino acid sequence alignment program Data indicated some amino acid homology (26% identity in 441 aa overlap of crtB gene of Neurospora crassa) with crtB genes of other organisms.

EXAMPLE 12 Cloning of the Phytoene Desaturase Gene (crtI) of Phaffia rhodozyma

a) Isolation of cDNA Clone

The entire library was excised into a phytoene accumulating cells of E. coli XL-Blue-MRF′, which carries the plasmid pACCAR25ΔcrtXΔcrtI (further indicated as XL-Blue-MRF′[pACCAR25ΔcrtXΔcrtI]). The screening for the crtI gene was based on the color of the transformants. Introduction of the crtI gene in a genetic background of XL-Blue-MRF′[pACCAR25ΔcrtXΔcrtI] would result in a restoration of the complete route for the biosynthesis of zeaxanthin, which could be monitored by the presence of a yellow/orange pigmented colony.

About 14.000 colonies were incubated on LB agar plates containing appropriate antibiotics and IPTG. Two colonies were found to have changed to a yellow/orange color.

b) Characterization of Complementing cDNA Clones

These colonies were streaked on LB-ampicillin agar plates. Plasmid DNA, designated pPRcrtI.1 and pPRcrtI.2, was isolated from these yellow colonies and found to include a 2.0 kb fragment (FIG. 2C). One of the resulting plasmids, pPRcrtI.1 was used for retransformation experiments (Table 8). Only the transformation of XL-Blue-MRF′[pACCAR25ΔcrtXΔcrtI] with pPRcrtI resulted in a white to yellow color change in phenotype. Therefore we tentative conclude that we have cloned a cDNA of P. rhodozyma encoding phytoene desaturase which is involved in the conversion of phytoene to lycopene.

TABLE 8 Color phenotype of carotenoid producing E. coli strains transformed with pPRcrtI. pUCI9 pPRcrtl XL-Blue-MRF′ white white (Ap, IPTG) XL-Blue-MRF′ white yellow/orange [pACCA25ΔcrtX ΔcrtI (Ap, Cm, IPTG) XL-Blue-MRF′ white white [pACCA25ΔcrtB (Ap, Cm, IPTG)

Legend: see Table 6.

c) Sequence Analysis of cDNA Fragment

One of the plasmid pPRcrtI was used to determine the nucleotide sequence of the 2.0 kb cDNA. The sequence comprised 2038 nucleotides and a 20 bp poly(A) tail. An open reading frame (ORF) of 582 amino acids was predicted. The nucleotide sequence and deduced amino acid sequence are shown in SEQIDNOs: 16 and 17, respectively. A search in SWISS-PROT protein sequence data bases using the Blitz amino acid sequence alignment program Data indicated amino acid homology to phytoene desaturase gene of N. crassa (53% identity in 529 aa overlap).

EXAMPLE 13 Cloning of the Lycopene Cyclase Gene (crtY) of Phaffia rhodozyma

a) Isolation of cDNA Clone

The entire library was excised into a lycopene accumulating cells of E. coli XL-Blue-MRF′, which carries the plasmid pACCRT-EIB (further indicated as XL-Blue-MRF′[pACCRT-EIB]). The screening for the crtY gene was based on the color of the transformant. Introduction of the crtY gene in a genetic background of XL-Blue-MRF′[pACCRT-EIB] would result in a restoration of the complete route for the biosynthesis of β-carotene, which could be monitored by the presence of a yellow pigmented colony. About 8.000 colonies were incubated on LB agar plates containing appropriate antibiotics and IPTG. One colony was found to have changed to a yellow color.

b) Characterization of Complementing cDNA Clone

This colony was streaked on LB-ampicillin agar plates. Plasmid DNA was isolated from this yellow colony and found to include a 2.5 kb fragment (FIG. 2B). The resulting plasmid, designated pPRcrtY, was used for retransformation experiments (Table 9. Surprisingly, not only transformation of XL-Blue-MRF′[pACCRT-EIB] but also transformation of XL-Blue-MRF′[pACCAR25ΔcrtB] with pPRcrtY resulted in a red to yellow color change in phenotype.

TABLE 9 Color phenotype of carotenoid producing E. coli strains transformed with pPRcrtY. pUC19 pPRcrtB XL-Blue-MRF′ white white (Ap, IPTG) XL-Blue-MRF′ red yellow [pACCRT-EIB (Ap, Cm, IPTG) XL-Blue-MRF′ red yellow [pACCA25ΔcrtB (Ap, Cm, IPTG)

Legend: see Table 6.

A second transformation experiment was carried out including the previously cloned cDNA of pPRcrtB. As shown in table 6 the cDNA previously (example 3) isolated as encoding phytoene synthase was able to complement the crtY deletion resulting in the biosynthesis of β-carotene in XL-Blue-MRF′[pACCRT-EIB].

Sequence analysis of the cDNA insert of pPRcrtY (SEQIDNOs: 18 and 19) showed that it was similar to the sequence of cDNA fragment of pPRcrtB.

From these data we tentative conclude that we have cloned a EDNA of P. rhodozyma encoding phytoene synthase and lycopene cyclase which is involved in the conversion of 2 GGPP molecules via prephytoene pyrophosphate into phytoene and lycopene to β-carotene, respectively. This is the first gene in a biosynthetic pathway of carotenoids synthesis that encodes two enzymatic activities.

TABLE 10 Color phenotype of carotenoid producing E. coli strains transformed with different cDNAs of Phaffia rhodozyma (Ap, Cm, IPTG). pUC19 pPRcrtE pPRcrtB pPRcrtY XL-Blue-MRF′ white yellow/ white white (pACCAR25ΔcrtE] orange XL-Blue-MRF′ white white yellow/ yellow/ [pACCA25ΔcrtB] orange orange XL-Blue-MRF′ red red yellow yellow [pACCRT-EIB]

Legend: see Table 6

EXAMPLE 14 Cloning of the Isopentenyl Diphosphate (IPP) Isomerase Gene (idi) of Phaffia rhodozyma

a) Isolation of cDNA Clone

The entire Phaffia cDNA library was excised into lycopene accumulating cells of E. coli XL-Blue-MRF′, each carrying the plasmid pACCRT-EIB (further indicated as XL-Blue-MRF′[pACCRT-EIB]). About 15.000 colonies were incubated on LB agar plates containing appropriate antibiotics and IPTG. One colony was found to have a dark red colour phenotype.

b) Characterization of Complementing cDNA Clone

This colony was streaked on LB-ampicillin agar plates. Plasmid DNA was isolated from this yellow colony and found to include a 1.1 kb fragment. The resulting plasmid, designated pPRcrtX, was used for retransformation experiments (Table 11).

All colonies of XL-Blue-MRF′[pACCAR-EIB] transformed with pPRcrtX had a dark red phenotype. From these data we tentatively concluded, that we have cloned a cDNA of P. rhodozyma expression of which results in an increased lycopene production in a genetically engineered E. coli strain.

TABLE 11 Color phenotype of carotenoid producing E. Coli strains transformed with pPRcrtX. pUC19 pPRcrtX XL-Blue-MRF′ white white (Ap, IPTG) XL-Blue-MRF′ red dark red [pACCRT-EIB (Ap Cm, IPTG)

Legend: see Table 6

c) Sequence Analysis of cDNA Fragment

It order to resolve the nature of this gene the complete nucleotide sequence of the cDNA insert in pPRcrtX was determined. The nucleotide sequence consist of the 1144 bp. The sequence comprised 1126 nucleotides and a poly(A) tail of 18 nucleotides. An open reading frame (ORF) of 251 aminoacids with a molecular mass of 28.7 kDa was predicted. The nucleotide sequence and deduced amino acid sequence are shown in SEQIDNOs: 20 and 21, respectively.

A search in SWISS-PROT protein sequence data bases using the Blitz amino acid sequence alignment program Data indicated aminoacid homology to isopentenyldiphosphate (IPP) isomerase (idi) of S. cerevisiae (42.2% identity in 200 aminoacid overlap). IPP isomerase catalyzes an essential activation step in the isoprene biosynthetic pathway which synthesis the 5-carbon building block of carotenoids. In analogy to yeast the gene of Phaffia was called idi1. The cDNA clone carrying the genes was then called pPRidi.

EXAMPLE 15 Overexpression of the idi Gene of P. rhodozyma in a Carotenogenic E. coli

Lycopene accumulating cells of E. coli XL-Blue-MRF′, which carry the plasmid pACCRT-EIB (further indicated as XL-Blue-MRF′[pACCRT-EIB]) were transformed with pUC19 and pPRidi and transformants were selected on solified LB-medium containing Amp and Cm. The transformants, called XL-Blue-MRF′[pACCRT-EIB/pUC19 and [pACCRT-EIB/pPRidi], were cultivated in 30 ml LB-medium containing Amp, Cm and IPTG at 3° C. at 250 rpm for 16 h. From these cultures 1 ml was used for carotenoid extraction and analysis. After centrifugation the cell pellet was dissolved in 200 μl aceton and incubated at 65°C. for 30 minutes. Fifty μl of the cell-free aceton fraction was then used for high-performance liquid chromatography (HPLC) analysis. The column (chrompack cat. 28265; packing nucleosil 100C18) was developed with water-acetonitrile-2-propanol (from 0 to 45 minutes 9:10:81 and after 45 minutes 2:18:80) at a flow rate of 0.4 ml per minute and recorded with a photodiode array detector at 470+/−20 nm. Lycopene was shown to have a retention time of about 23 minutes under these conditions. The peak area was used as the relative lycopene production (mAu*s). The relative lycopene production was 395 and 1165 for XL-Blue-MRF′[pACCRT-EIB/pUC19] and [pACCRT-EIB/pPRidi], respectively.

These data show the potentials of metabolic pathway engineering in Phaffia, as increased expression of the idi of Phaffia rhodozyma causes a 3-fold increase in carotenoid biosynthesis in E. coli.

This cDNA may be over-expressed in a transformed Phaffia cell with a view to enhance carotenoid and/or xanthophyll levels. The cDNA is suitably cloned under the control of a promoter active in Phaffia, such as a strong promoter according to his invention, for example a Phaffia glykolytic pathway promoter, such as the GAPDH-gene promoter disclosed herein, or a Phaffia ribosomal protein gene promoter according to the invention (vide sub). Optionally, the cDNA is cloned in front of a transcriptional terminator and/or polyadenylation site according to the invention, such as the GAPDH-gene terminator/polyadenylation site. The feasibility of this approach is illustrated in the next example, where the crtB gene from Erwinia uredovora is over-expressed in Phaffia rhodozyma by way of illustration.

EXAMPLE 16 Heterologous Expression of Carotenogenic Gene from Erwinia uredovora in Phaffia rhodozyma

The coding sequence encoding phytoene synthase (crtB) of Erwinia uredovora (Misawa et al., 1990) was cloned between the promoter and terminator sequences of the gpd (GAPDH-gene) of Phaffia by fusion PCR. In two separate PCR reactions the promoter sequence of gpd and the coding sequence of crtB were amplified. The former sequence was amplified using the primers 5177 and 5128 and pPR8 as template. This latter vector is a derivative of the Phaffia transformation vector pPR2 in which the promoter sequence has been enlarged and the BglII restriction site has been removed. The promoter sequence of gpd was amplified by PCR using the primers 5226 and 5307 and plasmid pPRgpd6 as template. The amplified promoter fragment was isolated, digested with KpnI and BamHI and cloned in the KpnI-BglII fragment of vector pPR2, yielding pPR8. The coding sequence of crtB was amplified using the primers 5131 and 5134 and pACCRT-EIB as template. In a second fusion PCR reaction, using the primers 5177 and 5134, 1 μg of the amplified promoter and crtB coding region fragment used as template yielding the fusion product Pgpd-crtB. The terminator sequence was amplified under standard PCR conditions using the primers 5137 and 5138 and the plasmid pPRgdh6 as template. Primer 5137 contains at the 5′ end the last 11 nucleotides of the coding region of the crtB gene of E. uredovora and the first 16 nucleotides of the terminator sequence of gpd gene of P. rhodozyma. By a two basepair substitution a BamHI restriction site was introduced. The amplified fusion product (Pgpd-crtB) and the amplified terminator fragments were purified and digested with HindIII and BamHI and cloned in the dephosphorylated HindIII site of the cloning vector pMTL25. The vector with the construct Pgpd-crtB-Tgpd was named pPREX1.1.

The HindIII fragment containing the expression cassette Pgpd-crtB-Tgpd was isolated from pPREX1.1 and ligated in the dephosphorylated HindIII site of the Phaffia transformation vector pPR8. After transformation of the ligation mixture into E. coli a vector (pPR8crtB6.1) with the correct insert was chosen for Phaffia transformation experiments.

Phaffia strain CBS6938 was transformed with pPRacrtB6.1, carrying the expression cassette Pgpd-crtB-Tgpd, and transformants were selected on plates containing G418. The relative amount of astaxanthin per OD₆₀₀ in three G418-resistant transformants and the wild-type Phaffia strains was determined by HPLC analysis (Table 12). For carotenoid isolation from Phaffia the method of DMSO/hexane extraction described by Sedmak et al., (1990; Biotechn. Techniq. 4, 107-112) was used.

TABLE 12 The relative astaxanthin production in a Phaffia transformant carrying the crtB gene of E. uredovora. Relative amount of astaxanthin Strain: (mAU*s/OD₆₆₀) P. rhodozyma CBS6938 448 P. rhodozyma CBS6938 [pPR8crtB6.1]#1 626 [pPR8crtB6.1]#2 716 [pPR8crtB6.1]#4 726 Primers used: 5128: 5′ caactgccat gatggtaagagtgttagag 3′ 5177: 5′ cccaagctttctcgag gtacctggtgggtgcatgtatgtac3′ 5131: 5′ taccatc atggcagttggctcgaaaag 3′ 5134: 5′ cccaagctt g ga t ccgt ctagagcgggcgctgcc3′ 5137: 5′ ccaaggcctaaacggatccctccaaacc 3′ 5138: 5′ gccaagcttctcgag cttgatcagataaagatagagat3′ 5307: 5′ gttgaagaagggatccttgtggatga 3′

The gpd sequences are indicated in bold, the crtB sequences in italic, additional restriction sites for cloning are underlined and base substitution are indicated by double underlining.

EXAMPLE 17 Isolation and Characterization of the crtB Gene of Phaffia

It will also be possible to express the Phaffia rhodozyma gene corresponding to crtB and express it under the control of its own regulatory regions, or under the control of a promoter of a highly expressed gene according ot the invention. The Phaffia transformation procedure disclosed herein, invariably leads to stably integrated high copy numbers of the introduced DNA, and it is expected, that expression of the gene under the control of its own promoter will also lead to enhanced production of carotenoids, including astaxanthin. To illustrate the principle, a protocol is given for the cloning of the crtB genomic sequence, below.

To obtain the genomic crtB-gene including expression signals the 2.5 kb BamHI-XhoI fragment was isolated from the vector pPRcrtB and used as probe to screen a cosmid library of Phaffia. The construction and screening of the library was carried out as described in Example 3 using the crtB gene as probe instead of the gapdh-gene.

After the rounds of hybridization, 2-colonies were identified giving a strong hybridization signal on the autoradiogram after exposure. Cosmid DNA isolated from these colonies was called pPRgcrtB#1.1 and pPRgcrtB#7, respectively.

Chromosomal DNA isolated from Phaffia rhodozyma strain CBS 6938 and cosmid pPRgcrtB#7 was digested with several restriction enzymes. The DNA fragments were separated, blotted and hybridized with a amino-terminal specific probe (0.45 kb XbaI fragment) of crtB under conditions as described before. After exposure, the autoradiogram showed DNA fragments of different length digested by different restriction enzymes which hybridized with the crtB probe. On the basis that no EcoRI site is present in the cDNA clone a EcoRI fragment of about 4.5 kb was chosen for subcloning experiments in order to determine the sequence in the promoter region and to establish the presence of intron sequences in the crtB gene. A similar sized hybridizing fragment was also found in the chromosomal DNA digested with EcoRI, The fragment was isolated from an agarose gel and ligated into the corresponding site of pUC19. The ligation mixture was transformed to competent E. coli cells. Plasmids with the correct insert in both orientations, named pPR10.1 and pPR10.2, were isolated from the transformants. Comparison of the restriction patterns of pPR10.1/pPR10.2 and pPRcrtB digested with XbaI gave an indication for the presence of one or more introns as the internal 2.0 kb XbaI fragment in the cDNA clone was found to be larger in the former vectors. The subclone pPR10.1 was used for sequence analysis of the promoter region and the structural gene by the so-called primer walking approach. The partial sequence of the insert in show in SEQIDNO: 22. Comparison of the cDNA and the genomic sequence revealed the presence of 4 introns.

EXAMPLE 18 Isolation of Promoter Sequences with High Expression Levels

This example illustrates the the feasibility of the “cDNA sequencing method” referred to in the detailed description, in order to obtain transcription promoters from highly expressed genes.

For the isolation and identification of transcription promoter sequences from Phaffia rhodozyma genes exhibiting high expression levels, the cDNA library of Phaffia rhodozyma was analyzed by the following procedure.

The cDNA library was plated on solified LB-medium containing Amp and 96 colonies were randomly picked for plasmid isolation. The purified plasmid was digested with XhoI and XbaI and loaded on a agarose gel. The size of the cDNA inserts varied from 0.5 to 3.0 kb. Subsequently, these plasmids were used as template for a single sequence reaction using the T3 primer. For 17 cDNA clones; no sequence data were obtained. The sequences obtained were translated in all three reading frames. For cDNA sequence the longest deduced amino acid sequences were compared with the SwissProt protein database at EBI using the Blitz program. For 18 deduced amino acid sequences no homology to n proteins was found whereas six amino acid sequences showed significant homology to hypothetical proteins. Fifty-five amino acid sequences were found to have significant homology to proteins for which the function is known. About 50% (38/79) were found to encode ribosomal proteins os which 12 full-length sequences were obtained.

TABLE 13 Overview of expressed cDNAs, encoded proteins and reference to the Sequence Listing cDNA coding for SEQIDNO: 10 ubiquitin-40S 24 11 Glu-repr.gene 26 18 40S rib.prot S27 28 35 60S rib.prot P1α 30 38 60S rib.prot L37e 32 46 605 rib.prot L27a 34 64 60S rib.prot L25 36 68 60S rib.prot P2 38 73 40S rib.prot S17A/B 40 76 40S rib.prot S31 42 78 40s rib.prot S10 44 85 60S rib.prot L37A 46 87 60S rib.prot L34 48 95 60S rib.prot S16 50

By sequence homology it was concluded that in Phaffia the 40S ribisomal protein S37 is fused to ubiquitin as is found in other organisms as well. The nucleotide sequences and deduced amino acid sequences of the full length cDNA clones are listed in the sequence listing. Six ribosomal proteins were represented in the random pool by more than one individual cDNA clone. The 40S ribosomal proteins S10 (SEQIDNO:44), S37 (+ ubiquitin) (SEQIDNO:24) and S27 (SEQIDNO:28) were represented twice and 60S (acidic) ribosomal proteins P2 (SEQIDNO:38), L37 (SEQIDNO:46) and L25 (SEQIDNO:36) found three times. From these results we conclude, that these proteins are encoded by multiple genes or that these genes are highly expressed. Therefore isolation of these promoter sequences are new and promissing target sequences to isolate high level expression signals from Phaffia rhodozyma. Furthermore, a cDNA clone was isolated which showed 50% homology to an abundant glucose-repressible gene from Neurospora crassa (Curr. genet. 14: 545-551 (1988)). The nucleotide sequence and the deduced amino acid sequence is shown in SEQIDNO:26. One of the advantages of such a promoter sequence is that it can be used to separated growth (biomass accumulation) and gene expression (product accumulation) in large scale Phaffia fermentation.

For the isolation of the promoter sequences of interest (as outlined above) a fragment from the corresponding cDNA clone can be used as probe to screen the genomic library of Phaffia rhodozyma following the approach as described for the GAPDH-gene promoter (Example 3, supra). Based on the determined nucleotide sequence of the promoter, specific oligonucleotides can be designed to construct a transcription fusion between the promoter and any gene of interest by the fusion PCR technique, following the procedure as outlined in Example 5 (supra).

EXAMPLE 19 Isolation of Carotenogenic Genes by Heterologous Hybridization

For the identification and isolation of corresponding carotenoid biosynthetic pathway genes from organisms related to Phaffia rhodozyma heterologous hybridization experiments were carried out under conditions of moderate stringency. In these experiments chromosomal DNA from two carotenogenic fungi (Neurospora crassa and Blakeslea trispora) and the yeasts S. cerevisiae and three yeast and fungal species from the genus Cystofylobasidium was used. These three carotenogenic yeasts are, based on phylogenetic studies, the ones most related to P. rhodozyma.

Chromosomal DNA from the yeast species Cystofylobasidium infirmo-miniatum (CBS 323), C. bisporidii (CBS 6346) and C. capitatum (CBS 6358) was isolated according the method as developed for Phaffia rhodozyma, described in example 3 of European patent application 0 590 707 A1; the relevant portions of which herein incorporated by reference. Isolation of chromosomal DNA from the fungi Neurospora crassa and Blakeslea trispora was essentially carried as described by Kolar et al. (Gene, 62: 127-134), the relevant parts of which are herein incorporated by reference.

Chromosomal DNA (5 μg) of C. infirmo-miniatum, C. bisporidii, C. capitatum, S. cerevisiae, P. rhodozyma, N. crassa and B. trispora was digested using EcoRI. The DNA fragments were separated on a 0.8% agarose gel, blotted and hybridized using the following conditions.

Hybridization was cared out at two temperatures (50° C. and 55° C.) using four different ³²P labelled Phaffia probes. The probes were made using random primed hexanucleotide labellings reactions using the XhoI-XbaI fragment(s) from the cDNA clones pPRcrtE, pPRcrtB, pPRcrtI and pPRidi as template. Hybridization was carried out o/n (16 h) at the indicated temperatures. After hybridization the filters were washed 2 times for 30 min. at the hybridization temperatures using a solution of 3*SSC; 0.1% SDS; 0.05% sodiumpyrophosphate. Films were developed after exposure of the filters to X-ray films in a cassette at −80° C. for 20 h.

Using the cDNA clone of crtE of P. rhodozyma faint signals were obtained for C. infirmo-miniatum, C. capitatum. Using the cDNA clone of crtB of P. rhodozyma strong signals were obtained to the high molecular weight portion of DNA from C. infirmo-miniatum and C. capitatum. Furthermore a strong signal was obtained in the lane loaded with digested chromosomal DNA from B. trispora. Only a faint signal was obtained for C. capitatum at 50° C. using the cDNA clone of crtI of P. rhodozyma. Using the cDNA clone of idi of P. rhodozyma faint signals were obtained with chromosomal DNA from C. infirmo-miniatum, C. bisporidii and C. capitatum at both temperatures. A strong signal was obtained in the lane loaded with digested chromosomal DNA from B. trispora.

We conclude, that carotenoid biosynthesis cDNAs or genes, or idi cDNAs or genes, can be isolated from other organisms, in particular from other yeast species by cross-hybridisation with the cDNA fragment coding for P. Rhodozyma carotenoid biosynthesis enzymes, or isopentenyl pyrophosphate isomerase coding sequences respectively, using moderately stringent hybridisation and washing conditions (50° C. to 55° C., 3×SSC).

Deposited Microorganisms

E. coli containing pGB-Ph9 has been deposited at the Centraal Bureau voor Schimmelcultures, Oosterstraat 1, Baarn, The Netherlands, on Jun. 23, 1993, under accession number CBS 359.3. The following strains have been deposited under the Budapest Treaty at the Centraal Bureau voor Schimmelcultures, Oosterstraat 1, Baarn, The Netherlands, on Feb. 26, 1996:

ID nr. Organism relevant feature Deposit number DS31855 E. coli crtY of P. rhodozyma CBS 232.96 DS31856 E. coli crtl of P. rhodozyma CBS 233.96 DS31857 E. coli crtE of P. rhodozyma CBS 234.96 DS31858 E. coli crtB of P. rhodozyma CBS 235.96

51 25 base pairs nucleic acid single linear DNA (genomic) NO not provided AB3005 1 CGGGATCCAA RCTNACNGGN ATGGC 25 32 base pairs nucleic acid single linear DNA (genomic) NO not provided AB3006 misc_feature one-of(12) /note= “N at position 12 is inosine” 2 CGGGATCCRT ANCCVYAYTC RTTRTCRTAC CA 32 27 base pairs nucleic acid single linear DNA (genomic) NO not provided AB4206 3 GCGTGACTTC TGGCCAGCCA CGATAGC 27 32 base pairs nucleic acid single linear DNA (genomic) NO not provided AB5126 4 TTCAATCCAC ATGATGGTAA GAGTGTTAGA GA 32 31 base pairs nucleic acid single linear DNA (genomic) NO not provided AB5127 5 CTTACCATCA TGTGGATTGA ACAAGATGGA T 31 40 base pairs nucleic acid single linear DNA (genomic) NO not provided AB5177 6 CCCAAGCTTC TCGAGGTACC TGGTGGGTGC ATGTATGTAC 40 30 base pairs nucleic acid single linear DNA (genomic) NO not provided AB5137 7 CCAAGGCCTA AAACGGATCC CTCCAAACCC 30 38 base pairs nucleic acid single linear DNA (genomic) NO not provided AB5138 8 GCCAAGCTTC TCGAGCTTGA TCAGATAAAG ATAGAGAT 38 2309 base pairs nucleic acid double linear DNA (genomic) NO NO Phaffia rhodozyma CBS 6938 exon 300..330 intron 331..530 exon 531..578 intron 579..668 exon 669..690 intron 691..767 exon 768..805 intron 806..905 exon 906..923 intron 924..1030 exon 1031..1378 intron 1379..1508 exon 1509..2020 CDS join(300..330, 531..578, 669..690, 768..805, 906 ..923, 1031..1378, 1509..2020) 9 GCTATGAGCA AGCACAACTG GGCACCGAAC GAGAACAGTA ACTGTCGGTA TCTTCCCACC 60 GACACGAGGC GTCTCCCGGC GGCAACCGCC GGTGCCCCCC TCCGCTTACG TCAGCCACCC 120 AGTTTTCTTC CATCTCTTTC TCTCTCCTTC CAAAAGTCTT TCAGTTTTAA ACGGCCCCCA 180 AAAAAAGAAG AGGCGACTTT TTCTTTCCTT CTCCCCATCA TCCACAAAGA TCTCTCTTCT 240 TCAACAACAA CTACTACTAC TACCACTACC ACCACTACTT CTCTAACACT CTTACCATC 299 ATG GCT GTC AAG GTT GGA ATC AAC GGT TTC G GTATGTGTTT GTTTTTCTCT 350 Met Ala Val Lys Val Gly Ile Asn Gly Phe 1 5 10 TGAGCTCCCC CATCGGTTCT TTCGCTTGTC CATGTTTCTT TTTCCTTTCC TTTCCTTTTC 410 TTTTTTCTCC CCACTGCCTT TTTTTTTTCT ATTCTTTTTT TTTTCCTTTC CTCTCGCCTT 470 CATGCATCGC ACTAACACCA TCTCATCTCA TCTCACTCTG CCTCGTCTTA CCTCCTACAG 530 GA CGA ATC GGA CGA ATC GTC CTT CGA AAC GCT ATC ATC CAC GGT GAT A 578 Gly Arg Ile Gly Arg Ile Val Leu Arg Asn Ala Ile Ile His Gly Asp 15 20 25 GTCAGTATTT TTTTAATTTC TTTTTTTCCC CATCAATTTC CCTCTGCTCC TTTACTCATC 638 TCTTTCCATC TCTCTCCCAC TCTCCTACAG TC GAT GTC GTC GCC ATC AAC GA 690 Ile Asp Val Val Ala Ile Asn Asp 30 GTGCGTCTAG ATCGACCATC TCGTCGTCCG CCCAAACACC GTCTGACACC ATCCTGTTAA 750 CTTTTCTCTC CTCCAAG C CCT TTC ATC GAT CTT GAG TAC ATG GTC TAC ATG 801 Pro Phe Ile Asp Leu Glu Tyr Met Val Tyr Met 35 40 45 TTC A GTAAGTCTCC CTCCCCCTCA AAAAGCCGAA ACAAAGCCGA ACAGAACCCG 855 Phe ATCTAACCAT TCGTTCTTCT TCCCTTTCCT CTTCCGTCTC TCCCTCACAG AG TAC 910 Lys Tyr GAC TCC ACC CAC G GTTCGTCCAT CCCTCTCTCT GTCCCGAACA TCTCCGACCG 963 Asp Ser Thr His 50 GGCCTTTTCC ATCTCCTGAT CCGTTCGCGT ACTAACCCAT ACCGTACCCT TCGTCCCATC 1023 CCTTCAG GT GTC TTC AAG GGA TCC GTC GAG ATC AAG GAC GGC AAG CTC 1071 Gly Val Phe Lys Gly Ser Val Glu Ile Lys Asp Gly Lys Leu 55 60 65 GTG ATC GAG GGC AAG CCC ATC GTC GTC TAC GGT GAG CGA GAC CCC GCC 1119 Val Ile Glu Gly Lys Pro Ile Val Val Tyr Gly Glu Arg Asp Pro Ala 70 75 80 AAC ATC CAG TGG GGA GCT GCC GGT GCC GAC TAC GTC GTC GAG TCC ACC 1167 Asn Ile Gln Trp Gly Ala Ala Gly Ala Asp Tyr Val Val Glu Ser Thr 85 90 95 GGT GTC TTC ACC ACC CAG GAG AAG GCC GAG CTC CAC CTC AAG GGA GGA 1215 Gly Val Phe Thr Thr Gln Glu Lys Ala Glu Leu His Leu Lys Gly Gly 100 105 110 GCC AAG AAG GTC GTC ATC TCT GCC CCT TCG GCC GAT GCC CCC ATG TTC 1263 Ala Lys Lys Val Val Ile Ser Ala Pro Ser Ala Asp Ala Pro Met Phe 115 120 125 130 GTC TGC GGT GTT AAC CTC GAC AAG TAC GAC CCC AAG TAC ACC GTC GTC 1311 Val Cys Gly Val Asn Leu Asp Lys Tyr Asp Pro Lys Tyr Thr Val Val 135 140 145 TCC AAC GCT TCG TGC ACC ACC AAC TGC TTG GCT CCC CTC GGC AAG GTC 1359 Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu Gly Lys Val 150 155 160 ATC CAC GAC AAC TAC ACC A GTCAGTCCTT TNCTTTGGAC TTGTCTGGCC 1408 Ile His Asp Asn Tyr Thr 165 TTTTCTTTGT TGGTTCTTTT CCTTTTGTCA AACCATCCAT ACTCACCCTG TTTTTCACCT 1468 TCTTTTTCTT CATTCACGTA TTCCCCCTCC CGTCCACCAG TT GTC GAG GGT CTC 1522 Ile Val Glu Gly Leu 170 ATG ACC ACC GTC CAC GCC ACC ACC GCC ACC CAG AAG ACC GTC GAC GGT 1570 Met Thr Thr Val His Ala Thr Thr Ala Thr Gln Lys Thr Val Asp Gly 175 180 185 CCT TCC AAC AAG GAC TGG CGA GGA GGT CGA GGA GCT GGT GCC AAC ATC 1618 Pro Ser Asn Lys Asp Trp Arg Gly Gly Arg Gly Ala Gly Ala Asn Ile 190 195 200 205 ATT CCC TCC TCC ACC GGA GCC GCC AAG GCC GTC GGT AAG GTT ATC CCC 1666 Ile Pro Ser Ser Thr Gly Ala Ala Lys Ala Val Gly Lys Val Ile Pro 210 215 220 TCC CTC AAC GGA AAG CTC ACC GGA ATG GCC TTC CGA GTG CCC ACC CCC 1714 Ser Leu Asn Gly Lys Leu Thr Gly Met Ala Phe Arg Val Pro Thr Pro 225 230 235 GAT GTC TCC GTC GTC GAT CTT GTC GTC CGA ATC GAG AAG GGC GCC TCT 1762 Asp Val Ser Val Val Asp Leu Val Val Arg Ile Glu Lys Gly Ala Ser 240 245 250 TAC GAG GAG ATC AAG GAG ACC ATC AAG AAG GCC TCC CAG ACC CCT GAG 1810 Tyr Glu Glu Ile Lys Glu Thr Ile Lys Lys Ala Ser Gln Thr Pro Glu 255 260 265 CTC AAG GGT ATC CTG AAC TAC ACC GAC GAC CAG GTC GTC TCC ACC GAT 1858 Leu Lys Gly Ile Leu Asn Tyr Thr Asp Asp Gln Val Val Ser Thr Asp 270 275 280 285 TTC ACC GGT GAC TCT GCC TCC TCC ACC TTC GAC GCC CAG GGC GGT ATC 1906 Phe Thr Gly Asp Ser Ala Ser Ser Thr Phe Asp Ala Gln Gly Gly Ile 290 295 300 TCC CTT AAC GGA AAC TTC GTC AAG CTT GTC TCC TGG TAC GAC AAC GAG 1954 Ser Leu Asn Gly Asn Phe Val Lys Leu Val Ser Trp Tyr Asp Asn Glu 305 310 315 TGG GGA TAC TCT GCC CGA GTC TGC GAC CTT GTT TCT TAC ATC GCC GCC 2002 Trp Gly Tyr Ser Ala Arg Val Cys Asp Leu Val Ser Tyr Ile Ala Ala 320 325 330 CAG GAC GCC AAG GCC TAAACGGTTC TCTCCAAACC CTCTCCCCTT TTGCCCTGCC 2057 Gln Asp Ala Lys Ala 335 CATTGAATTG ATTCCCTAAA TAGAATATCC CACTTTCTTT TATGCTCTAC CTATGATCAG 2117 TTTATCTGTC TTTTTCTTTG TGCGTGTCGG TTGTGCGACT GTACCCACCT CTTGAGGGAC 2177 AAGGCAAGAA GTGAGCAAGA TATGAACAAG AACAACAAAG AAAAAGAGAC AAAGAAAAAA 2237 AAAAGGAAAG AGAAAACAAT CCCCCCCCCC CCCCAAAAAA AAATCTCTAT CTTTATCTGA 2297 TCAAGAGATT AT 2309 338 amino acids amino acid linear protein not provided 10 Met Ala Val Lys Val Gly Ile Asn Gly Phe Gly Arg Ile Gly Arg Ile 1 5 10 15 Val Leu Arg Asn Ala Ile Ile His Gly Asp Ile Asp Val Val Ala Ile 20 25 30 Asn Asp Pro Phe Ile Asp Leu Glu Tyr Met Val Tyr Met Phe Lys Tyr 35 40 45 Asp Ser Thr His Gly Val Phe Lys Gly Ser Val Glu Ile Lys Asp Gly 50 55 60 Lys Leu Val Ile Glu Gly Lys Pro Ile Val Val Tyr Gly Glu Arg Asp 65 70 75 80 Pro Ala Asn Ile Gln Trp Gly Ala Ala Gly Ala Asp Tyr Val Val Glu 85 90 95 Ser Thr Gly Val Phe Thr Thr Gln Glu Lys Ala Glu Leu His Leu Lys 100 105 110 Gly Gly Ala Lys Lys Val Val Ile Ser Ala Pro Ser Ala Asp Ala Pro 115 120 125 Met Phe Val Cys Gly Val Asn Leu Asp Lys Tyr Asp Pro Lys Tyr Thr 130 135 140 Val Val Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu Gly 145 150 155 160 Lys Val Ile His Asp Asn Tyr Thr Ile Val Glu Gly Leu Met Thr Thr 165 170 175 Val His Ala Thr Thr Ala Thr Gln Lys Thr Val Asp Gly Pro Ser Asn 180 185 190 Lys Asp Trp Arg Gly Gly Arg Gly Ala Gly Ala Asn Ile Ile Pro Ser 195 200 205 Ser Thr Gly Ala Ala Lys Ala Val Gly Lys Val Ile Pro Ser Leu Asn 210 215 220 Gly Lys Leu Thr Gly Met Ala Phe Arg Val Pro Thr Pro Asp Val Ser 225 230 235 240 Val Val Asp Leu Val Val Arg Ile Glu Lys Gly Ala Ser Tyr Glu Glu 245 250 255 Ile Lys Glu Thr Ile Lys Lys Ala Ser Gln Thr Pro Glu Leu Lys Gly 260 265 270 Ile Leu Asn Tyr Thr Asp Asp Gln Val Val Ser Thr Asp Phe Thr Gly 275 280 285 Asp Ser Ala Ser Ser Thr Phe Asp Ala Gln Gly Gly Ile Ser Leu Asn 290 295 300 Gly Asn Phe Val Lys Leu Val Ser Trp Tyr Asp Asn Glu Trp Gly Tyr 305 310 315 320 Ser Ala Arg Val Cys Asp Leu Val Ser Tyr Ile Ala Ala Gln Asp Ala 325 330 335 Lys Ala 388 base pairs nucleic acid double linear DNA (genomic) NO NO Phaffia rhodozyma promoter 1..385 TATA_signal 249..263 /label= putative misc_signal 287..302 /function= “cap-signal” /label= putative misc_RNA 386..388 /function= “start of CDS” misc_feature 85 /note= “uncertain” 11 TGGTGGGTGC ATGTATGTAC GTGAGTGAGT GCGGGGGAAA GGCGAGTACG TGTGTGTACG 60 CGCAAGGAAG AACAACGAAG CGCANGCTAT GAGCAAGCAC AACTGGGCAC CGAACGAGAA 120 CAGTAACTGT CGGTATCTTC CCACCGACAC GAGGCGTCTC CCGGCGGCAA CCGCCGGTGC 180 CCCCCTCCGC TTACGTCAGC CACCCAGTTT TCTTCCATCT CTTTCTCTCT CCTTCCAAAA 240 GTCTTTCAGT TTTAAACGGC CCCCAAAAAA AGAAGAGGCG ACTTTTTCTT TCCTTCTCTC 300 CCATCATCCA CAAAGATCTC TCTTCTTCAA CAACAACTAC TACTACTACC ACTACCACCA 360 CTACTTCTCT AACACTCTTA CCATCATG 388 2546 base pairs nucleic acid double linear cDNA NO NO Phaffia rhodozyma CDS 225..2246 /product= “PRcrtB” 12 TCTAGAACTA GTGGATCCCC CGGGCTGCAG GAATTCGGCA CGAGCGGAAA CAAGAAGTGG 60 ACACAGAGAG ATCTTTGCTG AAGAGTTGTA TTCCAGAAAG GGAAAACAAA GGAAAGAAGC 120 GCCGAAGCAC ATCACCAACT TCAGCAAGCC GGTCCAGCCC GATCTCGGAT AGACATCATC 180 TTACCCAACT CGTATCATCC CCAACAGATA GAGTTTTTGT CGCA ATG ACG GCT CTC 236 Met Thr Ala Leu 1 GCA TAT TAC CAG ATC CAT CTG ATC TAT ACT CTC CCA ATT CTT GGT CTT 284 Ala Tyr Tyr Gln Ile His Leu Ile Tyr Thr Leu Pro Ile Leu Gly Leu 5 10 15 20 CTC GGC CTG CTC ACT TCC CCG ATT TTG ACA AAA TTT GAC ATC TAC AAA 332 Leu Gly Leu Leu Thr Ser Pro Ile Leu Thr Lys Phe Asp Ile Tyr Lys 25 30 35 ATA TCG ATC CTC GTA TTT ATT GCG TTT AGT GCA ACC ACA CCA TGG GAC 380 Ile Ser Ile Leu Val Phe Ile Ala Phe Ser Ala Thr Thr Pro Trp Asp 40 45 50 TCA TGG ATC ATC AGA AAT GGC GCA TGG ACA TAT CCA TCA GCG GAG AGT 428 Ser Trp Ile Ile Arg Asn Gly Ala Trp Thr Tyr Pro Ser Ala Glu Ser 55 60 65 GGC CAA GGC GTG TTT GGA ACG TTT CTA GAT GTT CCA TAT GAA GAG TAC 476 Gly Gln Gly Val Phe Gly Thr Phe Leu Asp Val Pro Tyr Glu Glu Tyr 70 75 80 GCT TTC TTT GTC ATT CAA ACC GTA ATC ACC GGC TTG GTC TAC GTC TTG 524 Ala Phe Phe Val Ile Gln Thr Val Ile Thr Gly Leu Val Tyr Val Leu 85 90 95 100 GCA ACT AGG CAC CTT CTC CCA TCT CTC GCG CTT CCC AAG ACT AGA TCG 572 Ala Thr Arg His Leu Leu Pro Ser Leu Ala Leu Pro Lys Thr Arg Ser 105 110 115 TCC GCC CTT TCT CTC GCG CTC AAG GCG CTC ATC CCT CTG CCC ATT ATC 620 Ser Ala Leu Ser Leu Ala Leu Lys Ala Leu Ile Pro Leu Pro Ile Ile 120 125 130 TAC CTA TTT ACC GCT CAC CCC AGC CCA TCG CCC GAC CCG CTC GTG ACA 668 Tyr Leu Phe Thr Ala His Pro Ser Pro Ser Pro Asp Pro Leu Val Thr 135 140 145 GAT CAC TAC TTC TAC ATG CGG GCA CTC TCC TTA CTC ATC ACC CCA CCT 716 Asp His Tyr Phe Tyr Met Arg Ala Leu Ser Leu Leu Ile Thr Pro Pro 150 155 160 ACC ATG CTC TTG GCA GCA TTA TCA GGC GAA TAT GCT TTC GAT TGG AAA 764 Thr Met Leu Leu Ala Ala Leu Ser Gly Glu Tyr Ala Phe Asp Trp Lys 165 170 175 180 AGT GGC CGA GCA AAG TCA ACT ATT GCA GCA ATC ATG ATC CCG ACG GTG 812 Ser Gly Arg Ala Lys Ser Thr Ile Ala Ala Ile Met Ile Pro Thr Val 185 190 195 TAT CTG ATT TGG GTA GAT TAT GTT GCT GTC GGT CAA GAC TCT TGG TCG 860 Tyr Leu Ile Trp Val Asp Tyr Val Ala Val Gly Gln Asp Ser Trp Ser 200 205 210 ATC AAC GAT GAG AAG ATT GTA GGG TGG AGG CTT GGA GGT GTA CTA CCC 908 Ile Asn Asp Glu Lys Ile Val Gly Trp Arg Leu Gly Gly Val Leu Pro 215 220 225 ATT GAG GAA GCT ATG TTC TTC TTA CTG ACG AAT CTA ATG ATT GTT CTG 956 Ile Glu Glu Ala Met Phe Phe Leu Leu Thr Asn Leu Met Ile Val Leu 230 235 240 GGT CTG TCT GCC TGC GAT CAT ACT CAG GCC CTA TAC CTG CTA CAC GGT 1004 Gly Leu Ser Ala Cys Asp His Thr Gln Ala Leu Tyr Leu Leu His Gly 245 250 255 260 CGA ACT ATT TAT GGC AAC AAA AAG ATG CCA TCT TCA TTT CCC CTC ATT 1052 Arg Thr Ile Tyr Gly Asn Lys Lys Met Pro Ser Ser Phe Pro Leu Ile 265 270 275 ACA CCG CCT GTG CTC TCC CTG TTT TTT AGC AGC CGA CCA TAC TCT TCT 1100 Thr Pro Pro Val Leu Ser Leu Phe Phe Ser Ser Arg Pro Tyr Ser Ser 280 285 290 CAG CCA AAA CGT GAC TTG GAA CTG GCA GTC AAG TTG TTG GAG AAA AAG 1148 Gln Pro Lys Arg Asp Leu Glu Leu Ala Val Lys Leu Leu Glu Lys Lys 295 300 305 AGC CGG AGC TTT TTT GTT GCC TCG GCT GGA TTT CCT AGC GAA GTT AGG 1196 Ser Arg Ser Phe Phe Val Ala Ser Ala Gly Phe Pro Ser Glu Val Arg 310 315 320 GAG AGG CTG GTT GGA CTA TAC GCA TTC TGC CGG GTG ACT GAT GAT CTT 1244 Glu Arg Leu Val Gly Leu Tyr Ala Phe Cys Arg Val Thr Asp Asp Leu 325 330 335 340 ATC GAC TCT CCT GAA GTA TCT TCC AAC CCG CAT GCC ACA ATT GAC ATG 1292 Ile Asp Ser Pro Glu Val Ser Ser Asn Pro His Ala Thr Ile Asp Met 345 350 355 GTC TCC GAT TTT CTT ACC CTA CTA TTT GGG CCC CCG CTA CAC CCT TCG 1340 Val Ser Asp Phe Leu Thr Leu Leu Phe Gly Pro Pro Leu His Pro Ser 360 365 370 CAA CCT GAC AAG ATC CTT TCT TCG CCT TTA CTT CCT CCT TCG CAC CCT 1388 Gln Pro Asp Lys Ile Leu Ser Ser Pro Leu Leu Pro Pro Ser His Pro 375 380 385 TCC CGA CCC ACG GGA ATG TAT CCC CTC CCG CCT CCT CCT TCG CTC TCG 1436 Ser Arg Pro Thr Gly Met Tyr Pro Leu Pro Pro Pro Pro Ser Leu Ser 390 395 400 CCT GCC GAG CTC GTT CAA TTC CTT ACC GAA AGG GTT CCC GTT CAA TAC 1484 Pro Ala Glu Leu Val Gln Phe Leu Thr Glu Arg Val Pro Val Gln Tyr 405 410 415 420 CAT TTC GCC TTC AGG TTG CTC GCT AAG TTG CAA GGG CTG ATC CCT CGA 1532 His Phe Ala Phe Arg Leu Leu Ala Lys Leu Gln Gly Leu Ile Pro Arg 425 430 435 TAC CCA CTC GAC GAA CTC CTT AGA GGA TAC ACC ACT GAT CTT ATC TTT 1580 Tyr Pro Leu Asp Glu Leu Leu Arg Gly Tyr Thr Thr Asp Leu Ile Phe 440 445 450 CCC TTA TCG ACA GAG GCA GTC CAG GCT CGG AAG ACG CCT ATC GAG ACC 1628 Pro Leu Ser Thr Glu Ala Val Gln Ala Arg Lys Thr Pro Ile Glu Thr 455 460 465 ACA GCT GAC TTG CTG GAC TAT GGT CTA TGT GTA GCA GGC TCA GTC GCC 1676 Thr Ala Asp Leu Leu Asp Tyr Gly Leu Cys Val Ala Gly Ser Val Ala 470 475 480 GAG CTA TTG GTC TAT GTC TCT TGG GCA AGT GCA CCA AGT CAG GTC CCT 1724 Glu Leu Leu Val Tyr Val Ser Trp Ala Ser Ala Pro Ser Gln Val Pro 485 490 495 500 GCC ACC ATA GAA GAA AGA GAA GCT GTG TTA GTG GCA AGC CGA GAG ATG 1772 Ala Thr Ile Glu Glu Arg Glu Ala Val Leu Val Ala Ser Arg Glu Met 505 510 515 GGA ACT GCC CTT CAG TTG GTG AAC ATT GCT AGG GAC ATT AAA GGG GAC 1820 Gly Thr Ala Leu Gln Leu Val Asn Ile Ala Arg Asp Ile Lys Gly Asp 520 525 530 GCA ACA GAA GGG AGA TTT TAC CTA CCA CTC TCA TTC TTT GGT CTT CGG 1868 Ala Thr Glu Gly Arg Phe Tyr Leu Pro Leu Ser Phe Phe Gly Leu Arg 535 540 545 GAT GAA TCA AAG CTT GCG ATC CCG ACT GAT TGG ACG GAA CCT CGG CCT 1916 Asp Glu Ser Lys Leu Ala Ile Pro Thr Asp Trp Thr Glu Pro Arg Pro 550 555 560 CAA GAT TTC GAC AAA CTC CTC AGT CTA TCT CCT TCG TCC ACA TTA CCA 1964 Gln Asp Phe Asp Lys Leu Leu Ser Leu Ser Pro Ser Ser Thr Leu Pro 565 570 575 580 TCT TCA AAC GCC TCA GAA AGC TTC CGG TTC GAA TGG AAG ACG TAC TCG 2012 Ser Ser Asn Ala Ser Glu Ser Phe Arg Phe Glu Trp Lys Thr Tyr Ser 585 590 595 CTT CCA TTA GTC GCC TAC GCA GAG GAT CTT GCC AAA CAT TCT TAT AAG 2060 Leu Pro Leu Val Ala Tyr Ala Glu Asp Leu Ala Lys His Ser Tyr Lys 600 605 610 GGA ATT GAC CGA CTT CCT ACC GAG GTT CAA GCG GGA ATG CGA GCG GCT 2108 Gly Ile Asp Arg Leu Pro Thr Glu Val Gln Ala Gly Met Arg Ala Ala 615 620 625 TGC GCG AGC TAC CTA CTG ATC GGC CGA GAG ATC AAA GTC GTT TGG AAA 2156 Cys Ala Ser Tyr Leu Leu Ile Gly Arg Glu Ile Lys Val Val Trp Lys 630 635 640 GGA GAC GTC GGA GAG AGA AGG ACA GTT GCC GGA TGG AGG AGA GTA CGG 2204 Gly Asp Val Gly Glu Arg Arg Thr Val Ala Gly Trp Arg Arg Val Arg 645 650 655 660 AAA GTC TTG AGT GTG GTC ATG AGC GGA TGG GAA GGG CAG TAAGACAGCG 2253 Lys Val Leu Ser Val Val Met Ser Gly Trp Glu Gly Gln 665 670 GAAGAATACC GACAGACAAT GATGAGTGAG AATAAAATCA TCCTCAATCT TCTTTCTCTA 2313 GGTGCTCTTT TTTGTTTTCT ATTATGACCA ACTCTAAAGG AACTGGCCTT GCAGATATTT 2373 CTCTTCCCCC CATCTTCCTC CTTTCCATCG TTTGTTCTTT CCATTTTTGT CGGTTTACTA 2433 TGTCAATTCT TTTTCTTGCT TTTTCTTATC AATCTAGACA ATTCTATAGA TGTTTAGAAT 2493 TTATACATTG ACAGGTTATA GACCATAAAG ACTAAAAAAA AAAAAAAAAA AAA 2546 673 amino acids amino acid linear protein not provided 13 Met Thr Ala Leu Ala Tyr Tyr Gln Ile His Leu Ile Tyr Thr Leu Pro 1 5 10 15 Ile Leu Gly Leu Leu Gly Leu Leu Thr Ser Pro Ile Leu Thr Lys Phe 20 25 30 Asp Ile Tyr Lys Ile Ser Ile Leu Val Phe Ile Ala Phe Ser Ala Thr 35 40 45 Thr Pro Trp Asp Ser Trp Ile Ile Arg Asn Gly Ala Trp Thr Tyr Pro 50 55 60 Ser Ala Glu Ser Gly Gln Gly Val Phe Gly Thr Phe Leu Asp Val Pro 65 70 75 80 Tyr Glu Glu Tyr Ala Phe Phe Val Ile Gln Thr Val Ile Thr Gly Leu 85 90 95 Val Tyr Val Leu Ala Thr Arg His Leu Leu Pro Ser Leu Ala Leu Pro 100 105 110 Lys Thr Arg Ser Ser Ala Leu Ser Leu Ala Leu Lys Ala Leu Ile Pro 115 120 125 Leu Pro Ile Ile Tyr Leu Phe Thr Ala His Pro Ser Pro Ser Pro Asp 130 135 140 Pro Leu Val Thr Asp His Tyr Phe Tyr Met Arg Ala Leu Ser Leu Leu 145 150 155 160 Ile Thr Pro Pro Thr Met Leu Leu Ala Ala Leu Ser Gly Glu Tyr Ala 165 170 175 Phe Asp Trp Lys Ser Gly Arg Ala Lys Ser Thr Ile Ala Ala Ile Met 180 185 190 Ile Pro Thr Val Tyr Leu Ile Trp Val Asp Tyr Val Ala Val Gly Gln 195 200 205 Asp Ser Trp Ser Ile Asn Asp Glu Lys Ile Val Gly Trp Arg Leu Gly 210 215 220 Gly Val Leu Pro Ile Glu Glu Ala Met Phe Phe Leu Leu Thr Asn Leu 225 230 235 240 Met Ile Val Leu Gly Leu Ser Ala Cys Asp His Thr Gln Ala Leu Tyr 245 250 255 Leu Leu His Gly Arg Thr Ile Tyr Gly Asn Lys Lys Met Pro Ser Ser 260 265 270 Phe Pro Leu Ile Thr Pro Pro Val Leu Ser Leu Phe Phe Ser Ser Arg 275 280 285 Pro Tyr Ser Ser Gln Pro Lys Arg Asp Leu Glu Leu Ala Val Lys Leu 290 295 300 Leu Glu Lys Lys Ser Arg Ser Phe Phe Val Ala Ser Ala Gly Phe Pro 305 310 315 320 Ser Glu Val Arg Glu Arg Leu Val Gly Leu Tyr Ala Phe Cys Arg Val 325 330 335 Thr Asp Asp Leu Ile Asp Ser Pro Glu Val Ser Ser Asn Pro His Ala 340 345 350 Thr Ile Asp Met Val Ser Asp Phe Leu Thr Leu Leu Phe Gly Pro Pro 355 360 365 Leu His Pro Ser Gln Pro Asp Lys Ile Leu Ser Ser Pro Leu Leu Pro 370 375 380 Pro Ser His Pro Ser Arg Pro Thr Gly Met Tyr Pro Leu Pro Pro Pro 385 390 395 400 Pro Ser Leu Ser Pro Ala Glu Leu Val Gln Phe Leu Thr Glu Arg Val 405 410 415 Pro Val Gln Tyr His Phe Ala Phe Arg Leu Leu Ala Lys Leu Gln Gly 420 425 430 Leu Ile Pro Arg Tyr Pro Leu Asp Glu Leu Leu Arg Gly Tyr Thr Thr 435 440 445 Asp Leu Ile Phe Pro Leu Ser Thr Glu Ala Val Gln Ala Arg Lys Thr 450 455 460 Pro Ile Glu Thr Thr Ala Asp Leu Leu Asp Tyr Gly Leu Cys Val Ala 465 470 475 480 Gly Ser Val Ala Glu Leu Leu Val Tyr Val Ser Trp Ala Ser Ala Pro 485 490 495 Ser Gln Val Pro Ala Thr Ile Glu Glu Arg Glu Ala Val Leu Val Ala 500 505 510 Ser Arg Glu Met Gly Thr Ala Leu Gln Leu Val Asn Ile Ala Arg Asp 515 520 525 Ile Lys Gly Asp Ala Thr Glu Gly Arg Phe Tyr Leu Pro Leu Ser Phe 530 535 540 Phe Gly Leu Arg Asp Glu Ser Lys Leu Ala Ile Pro Thr Asp Trp Thr 545 550 555 560 Glu Pro Arg Pro Gln Asp Phe Asp Lys Leu Leu Ser Leu Ser Pro Ser 565 570 575 Ser Thr Leu Pro Ser Ser Asn Ala Ser Glu Ser Phe Arg Phe Glu Trp 580 585 590 Lys Thr Tyr Ser Leu Pro Leu Val Ala Tyr Ala Glu Asp Leu Ala Lys 595 600 605 His Ser Tyr Lys Gly Ile Asp Arg Leu Pro Thr Glu Val Gln Ala Gly 610 615 620 Met Arg Ala Ala Cys Ala Ser Tyr Leu Leu Ile Gly Arg Glu Ile Lys 625 630 635 640 Val Val Trp Lys Gly Asp Val Gly Glu Arg Arg Thr Val Ala Gly Trp 645 650 655 Arg Arg Val Arg Lys Val Leu Ser Val Val Met Ser Gly Trp Glu Gly 660 665 670 Gln 1882 base pairs nucleic acid double linear cDNA NO NO Phaffia rhodozyma CDS 82..1212 /product= “PRcrtE” 14 GGCACGAGCC AATTTAAAGT GCACTCAGCC ATAGCTAACA CACAGAACTA CACATACATA 60 CACTCATCCG GAACACATAG G ATG GAT TAC GCG AAC ATC CTC ACA GCA ATT 111 Met Asp Tyr Ala Asn Ile Leu Thr Ala Ile 1 5 10 CCA CTC GAG TTT ACT CCT CAG GAT GAT ATC GTG CTC CTT GAA CCG TAT 159 Pro Leu Glu Phe Thr Pro Gln Asp Asp Ile Val Leu Leu Glu Pro Tyr 15 20 25 CAC TAC CTA GGA AAG AAC CCT GGA AAA GAA ATT CGA TCA CAA CTC ATC 207 His Tyr Leu Gly Lys Asn Pro Gly Lys Glu Ile Arg Ser Gln Leu Ile 30 35 40 GAG GCT TTC AAC TAT TGG TTG GAT GTC AAG AAG GAG GAT CTC GAG GTC 255 Glu Ala Phe Asn Tyr Trp Leu Asp Val Lys Lys Glu Asp Leu Glu Val 45 50 55 ATC CAG AAC GTT GTT GGC ATG CTA CAT ACC GCT AGC TTA TTA ATG GAC 303 Ile Gln Asn Val Val Gly Met Leu His Thr Ala Ser Leu Leu Met Asp 60 65 70 GAT GTG GAG GAT TCA TCG GTC CTC AGG CGT GGG TCG CCT GTG GCC CAT 351 Asp Val Glu Asp Ser Ser Val Leu Arg Arg Gly Ser Pro Val Ala His 75 80 85 90 CTA ATT TAC GGG ATT CCG CAG ACA ATA AAC ACT GCA AAC TAC GTC TAC 399 Leu Ile Tyr Gly Ile Pro Gln Thr Ile Asn Thr Ala Asn Tyr Val Tyr 95 100 105 TTT CTG GCT TAT CAA GAG ATC TTC AAG CTT CGC CCA ACA CCG ATA CCC 447 Phe Leu Ala Tyr Gln Glu Ile Phe Lys Leu Arg Pro Thr Pro Ile Pro 110 115 120 ATG CCT GTA ATT CCT CCT TCA TCT GCT TCG CTT CAA TCA TCC GTC TCC 495 Met Pro Val Ile Pro Pro Ser Ser Ala Ser Leu Gln Ser Ser Val Ser 125 130 135 TCT GCA TCC TCC TCC TCC TCG GCC TCG TCT GAA AAC GGG GGC ACG TCA 543 Ser Ala Ser Ser Ser Ser Ser Ala Ser Ser Glu Asn Gly Gly Thr Ser 140 145 150 ACT CCT AAT TCG CAG ATT CCG TTC TCG AAA GAT ACG TAT CTT GAT AAA 591 Thr Pro Asn Ser Gln Ile Pro Phe Ser Lys Asp Thr Tyr Leu Asp Lys 155 160 165 170 GTG ATC ACA GAC GAG ATG CTT TCC CTC CAT AGA GGG CAA GGC CTG GAG 639 Val Ile Thr Asp Glu Met Leu Ser Leu His Arg Gly Gln Gly Leu Glu 175 180 185 CTA TTC TGG AGA GAT AGT CTG ACG TGT CCT AGC GAA GAG GAA TAT GTG 687 Leu Phe Trp Arg Asp Ser Leu Thr Cys Pro Ser Glu Glu Glu Tyr Val 190 195 200 AAA ATG GTT CTT GGA AAG ACG GGA GGT TTG TTC CGT ATA GCG GTC AGA 735 Lys Met Val Leu Gly Lys Thr Gly Gly Leu Phe Arg Ile Ala Val Arg 205 210 215 TTG ATG ATG GCA AAG TCA GAA TGT GAC ATA GAC TTT GTC CAG CTT GTC 783 Leu Met Met Ala Lys Ser Glu Cys Asp Ile Asp Phe Val Gln Leu Val 220 225 230 AAC TTG ATC TCA ATA TAC TTC CAG ATC AGG GAT GAC TAT ATG AAC CTT 831 Asn Leu Ile Ser Ile Tyr Phe Gln Ile Arg Asp Asp Tyr Met Asn Leu 235 240 245 250 CAG TCT TCT GAG TAT GCC CAT AAT AAG AAT TTT GCA GAG GAC CTC ACA 879 Gln Ser Ser Glu Tyr Ala His Asn Lys Asn Phe Ala Glu Asp Leu Thr 255 260 265 GAA GGG AAA TTC AGT TTT CCC ACT ATC CAC TCG ATT CAT GCC AAC CCC 927 Glu Gly Lys Phe Ser Phe Pro Thr Ile His Ser Ile His Ala Asn Pro 270 275 280 TCA TCG AGA CTC GTC ATC AAT ACG TTG CAG AAG AAA TCG ACC TCT CCT 975 Ser Ser Arg Leu Val Ile Asn Thr Leu Gln Lys Lys Ser Thr Ser Pro 285 290 295 GAG ATC CTT CAC CAC TGT GTA AAC TAC ATG CGC ACA GAA ACC CAC TCA 1023 Glu Ile Leu His His Cys Val Asn Tyr Met Arg Thr Glu Thr His Ser 300 305 310 TTC GAA TAT ACT CAG GAA GTC CTC AAC ACC TTG TCA GGT GCA CTC GAG 1071 Phe Glu Tyr Thr Gln Glu Val Leu Asn Thr Leu Ser Gly Ala Leu Glu 315 320 325 330 AGA GAA CTA GGA AGG CTT CAA GGA GAG TTC GCA GAA GCT AAC TCA AGG 1119 Arg Glu Leu Gly Arg Leu Gln Gly Glu Phe Ala Glu Ala Asn Ser Arg 335 340 345 ATG GAT CTT GGA GAC GTA GAT TCG GAA GGA AGA ACG GGG AAG AAC GTC 1167 Met Asp Leu Gly Asp Val Asp Ser Glu Gly Arg Thr Gly Lys Asn Val 350 355 360 AAA TTG GAA GCG ATC CTG AAA AAG CTA GCC GAT ATC CCT CTG TGAAAGAACA 1219 Lys Leu Glu Ala Ile Leu Lys Lys Leu Ala Asp Ile Pro Leu 365 370 375 TATTCTCTCT CTCGTCTGTC CGTTTCTATC AGGGTTTTAT AAGTTGTCTC TTTATTCCTA 1279 AGGGTTTGTC AGATGATTGG ACTTGATGTG CTCTATTGCC CGTTCATCTT TTTCACTTCG 1339 ACTTTTTTCT CTACCGTGCA TGCCCATTCG CATTCTCTTG TTCATCTTGT GTTTAATTTG 1399 TTCGACATAA CATTAATCAT CGTGTCTTCT TCTTTTCGAA GAAATCTCGT GACTTGTTGA 1459 ACTTCAACTA TAATTAATCA TATTCATATC TCAAAGTCTT CGTCTTCTCG CAATGTGATT 1519 CCTCCTTCCA GTTCCCTCTT TGATTTCCTT CTCATTGATC GGTTTCTTTT TCTTTTTTGC 1579 TCTCCTGTCT CTTCTTTATT CGCCTTCCGT CTCTCTGTCT CGTTTTCTCT TCACTTTTTT 1639 TTTTCATCTT CTCTCGGTCA ACTTGTCATT TAATCTCTCT AGGGTCTCAT GTCAACACGT 1699 GCCAAGCATG TCATACGTGT GCAGGGTGAT GTACAGTCAT TTTGCCATCC CTCTTCGCAG 1759 GGTCTCATCT ATCTTGTCTA TCGACTTTTC CTCTTTTTGA ATTTCCTCGG AGTTTTATCT 1819 TGGTATAAGC AATGGAGAAG AGCGCAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAACTCG 1879 AGG 1882 376 amino acids amino acid linear protein not provided 15 Met Asp Tyr Ala Asn Ile Leu Thr Ala Ile Pro Leu Glu Phe Thr Pro 1 5 10 15 Gln Asp Asp Ile Val Leu Leu Glu Pro Tyr His Tyr Leu Gly Lys Asn 20 25 30 Pro Gly Lys Glu Ile Arg Ser Gln Leu Ile Glu Ala Phe Asn Tyr Trp 35 40 45 Leu Asp Val Lys Lys Glu Asp Leu Glu Val Ile Gln Asn Val Val Gly 50 55 60 Met Leu His Thr Ala Ser Leu Leu Met Asp Asp Val Glu Asp Ser Ser 65 70 75 80 Val Leu Arg Arg Gly Ser Pro Val Ala His Leu Ile Tyr Gly Ile Pro 85 90 95 Gln Thr Ile Asn Thr Ala Asn Tyr Val Tyr Phe Leu Ala Tyr Gln Glu 100 105 110 Ile Phe Lys Leu Arg Pro Thr Pro Ile Pro Met Pro Val Ile Pro Pro 115 120 125 Ser Ser Ala Ser Leu Gln Ser Ser Val Ser Ser Ala Ser Ser Ser Ser 130 135 140 Ser Ala Ser Ser Glu Asn Gly Gly Thr Ser Thr Pro Asn Ser Gln Ile 145 150 155 160 Pro Phe Ser Lys Asp Thr Tyr Leu Asp Lys Val Ile Thr Asp Glu Met 165 170 175 Leu Ser Leu His Arg Gly Gln Gly Leu Glu Leu Phe Trp Arg Asp Ser 180 185 190 Leu Thr Cys Pro Ser Glu Glu Glu Tyr Val Lys Met Val Leu Gly Lys 195 200 205 Thr Gly Gly Leu Phe Arg Ile Ala Val Arg Leu Met Met Ala Lys Ser 210 215 220 Glu Cys Asp Ile Asp Phe Val Gln Leu Val Asn Leu Ile Ser Ile Tyr 225 230 235 240 Phe Gln Ile Arg Asp Asp Tyr Met Asn Leu Gln Ser Ser Glu Tyr Ala 245 250 255 His Asn Lys Asn Phe Ala Glu Asp Leu Thr Glu Gly Lys Phe Ser Phe 260 265 270 Pro Thr Ile His Ser Ile His Ala Asn Pro Ser Ser Arg Leu Val Ile 275 280 285 Asn Thr Leu Gln Lys Lys Ser Thr Ser Pro Glu Ile Leu His His Cys 290 295 300 Val Asn Tyr Met Arg Thr Glu Thr His Ser Phe Glu Tyr Thr Gln Glu 305 310 315 320 Val Leu Asn Thr Leu Ser Gly Ala Leu Glu Arg Glu Leu Gly Arg Leu 325 330 335 Gln Gly Glu Phe Ala Glu Ala Asn Ser Arg Met Asp Leu Gly Asp Val 340 345 350 Asp Ser Glu Gly Arg Thr Gly Lys Asn Val Lys Leu Glu Ala Ile Leu 355 360 365 Lys Lys Leu Ala Asp Ile Pro Leu 370 375 2058 base pairs nucleic acid double linear cDNA NO NO Phaffia rhodozyma CDS 46..1794 /product= “PRcrtI” 16 CCTCGCCGAA TCTAACTTGA CACATAACTC TAGTATCTAT ACTCG ATG GGA AAA 54 Met Gly Lys 1 GAA CAA GAT CAG GAT AAA CCC ACA GCT ATC ATC GTG GGA TGT GGT ATC 102 Glu Gln Asp Gln Asp Lys Pro Thr Ala Ile Ile Val Gly Cys Gly Ile 5 10 15 GGT GGA ATC GCC ACT GCC GCT CGT CTT GCT AAA GAA GGT TTC CAG GTC 150 Gly Gly Ile Ala Thr Ala Ala Arg Leu Ala Lys Glu Gly Phe Gln Val 20 25 30 35 ACG GTG TTC GAG AAG AAC GAC TAC TCC GGA GGT CGA TGC TCT TTA ATC 198 Thr Val Phe Glu Lys Asn Asp Tyr Ser Gly Gly Arg Cys Ser Leu Ile 40 45 50 GAG CGA GAT GGT TAT CGA TTC GAT CAG GGG CCC AGT TTG CTG CTC TTG 246 Glu Arg Asp Gly Tyr Arg Phe Asp Gln Gly Pro Ser Leu Leu Leu Leu 55 60 65 CCA GAT CTC TTC AAG CAG ACA TTC GAA GAT TTG GGA GAG AAG ATG GAA 294 Pro Asp Leu Phe Lys Gln Thr Phe Glu Asp Leu Gly Glu Lys Met Glu 70 75 80 GAT TGG GTC GAT CTC ATC AAG TGT GAA CCC AAC TAT GTT TGC CAC TTC 342 Asp Trp Val Asp Leu Ile Lys Cys Glu Pro Asn Tyr Val Cys His Phe 85 90 95 CAC GAT GAA GAG ACT TTC ACT TTT TCA ACC GAC ATG GCG TTG CTC AAG 390 His Asp Glu Glu Thr Phe Thr Phe Ser Thr Asp Met Ala Leu Leu Lys 100 105 110 115 CGG GAA GTC GAG CGT TTT GAA GGC AAA GAT GGA TTT GAT CGG TTC TTG 438 Arg Glu Val Glu Arg Phe Glu Gly Lys Asp Gly Phe Asp Arg Phe Leu 120 125 130 TCG TTT ATC CAA GAA GCC CAC AGA CAT TAC GAG CTT GCT GTC GTT CAC 486 Ser Phe Ile Gln Glu Ala His Arg His Tyr Glu Leu Ala Val Val His 135 140 145 GTC CTG CAG AAG AAC TTC CCT GGC TTC GCA GCA TTC TTA CGG CTA CAG 534 Val Leu Gln Lys Asn Phe Pro Gly Phe Ala Ala Phe Leu Arg Leu Gln 150 155 160 TTC ATT GGC CAA ATC CTG GCT CTT CAC CCC TTC GAG TCT ATC TGG ACA 582 Phe Ile Gly Gln Ile Leu Ala Leu His Pro Phe Glu Ser Ile Trp Thr 165 170 175 AGA GTT TGT CGA TAT TTC AAG ACC GAC AGA TTA CGA AGA GTC TTC TCG 630 Arg Val Cys Arg Tyr Phe Lys Thr Asp Arg Leu Arg Arg Val Phe Ser 180 185 190 195 TTT GCA GTG ATG TAC ATG GGT CAA AGC CCA TAC AGT GCG CCC GGA ACA 678 Phe Ala Val Met Tyr Met Gly Gln Ser Pro Tyr Ser Ala Pro Gly Thr 200 205 210 TAT TCC TTG CTC CAA TAC ACC GAA TTG ACC GAG GGC ATC TGG TAT CCG 726 Tyr Ser Leu Leu Gln Tyr Thr Glu Leu Thr Glu Gly Ile Trp Tyr Pro 215 220 225 AGA GGA GGC TTT TGG CAG GTT CCT AAT ACT CTT CTT CAG ATC GTC AAG 774 Arg Gly Gly Phe Trp Gln Val Pro Asn Thr Leu Leu Gln Ile Val Lys 230 235 240 CGC AAC AAT CCC TCA GCC AAG TTC AAT TTC AAC GCT CCA GTT TCC CAG 822 Arg Asn Asn Pro Ser Ala Lys Phe Asn Phe Asn Ala Pro Val Ser Gln 245 250 255 GTT CTT CTC TCT CCT GCC AAG GAC CGA GCG ACT GGT GTT CGA CTT GAA 870 Val Leu Leu Ser Pro Ala Lys Asp Arg Ala Thr Gly Val Arg Leu Glu 260 265 270 275 TCC GGC GAG GAA CAT CAC GCC GAT GTT GTG ATT GTC AAT GCT GAC CTC 918 Ser Gly Glu Glu His His Ala Asp Val Val Ile Val Asn Ala Asp Leu 280 285 290 GTT TAC GCC TCC GAG CAC TTG ATT CCT GAC GAT GCC AGA AAC AAG ATT 966 Val Tyr Ala Ser Glu His Leu Ile Pro Asp Asp Ala Arg Asn Lys Ile 295 300 305 GGC CAA CTG GGT GAA GTC AAG AGA AGT TGG TGG GCT GAC TTA GTT GGT 1014 Gly Gln Leu Gly Glu Val Lys Arg Ser Trp Trp Ala Asp Leu Val Gly 310 315 320 GGA AAG AAG CTC AAG GGA AGT TGC AGT AGT TTG AGC TTC TAC TGG AGC 1062 Gly Lys Lys Leu Lys Gly Ser Cys Ser Ser Leu Ser Phe Tyr Trp Ser 325 330 335 ATG GAC CGA ATC GTG GAC GGT CTG GGC GGA CAC AAT ATC TTC TTG GCC 1110 Met Asp Arg Ile Val Asp Gly Leu Gly Gly His Asn Ile Phe Leu Ala 340 345 350 355 GAG GAC TTC AAG GGA TCA TTC GAC ACA ATC TTC GAG GAG TTG GGT CTC 1158 Glu Asp Phe Lys Gly Ser Phe Asp Thr Ile Phe Glu Glu Leu Gly Leu 360 365 370 CCA GCC GAT CCT TCC TTT TAC GTG AAC GTT CCC TCG CGA ATC GAT CCT 1206 Pro Ala Asp Pro Ser Phe Tyr Val Asn Val Pro Ser Arg Ile Asp Pro 375 380 385 TCT GCC GCT CCC GAA GGC AAA GAT GCT ATC GTC ATT CTT GTG CCG TGT 1254 Ser Ala Ala Pro Glu Gly Lys Asp Ala Ile Val Ile Leu Val Pro Cys 390 395 400 GGC CAT ATC GAC GCT TCG AAC CCT CAA GAT TAC AAC AAG CTT GTT GCT 1302 Gly His Ile Asp Ala Ser Asn Pro Gln Asp Tyr Asn Lys Leu Val Ala 405 410 415 CGG GCA AGG AAG TTT GTG ATC CAA ACG CTT TCC GCC AAG CTT GGA CTT 1350 Arg Ala Arg Lys Phe Val Ile Gln Thr Leu Ser Ala Lys Leu Gly Leu 420 425 430 435 CCC GAC TTT GAA AAA ATG ATT GTG GCA GAG AAG GTT CAC GAT GCT CCC 1398 Pro Asp Phe Glu Lys Met Ile Val Ala Glu Lys Val His Asp Ala Pro 440 445 450 TCT TGG GAG AAA GAA TTT AAC CTC AAG GAC GGA AGC ATC TTG GGA CTG 1446 Ser Trp Glu Lys Glu Phe Asn Leu Lys Asp Gly Ser Ile Leu Gly Leu 455 460 465 GCT CAC AAC TTT ATG CAA GTT CTT GGT TTC AGG CCG AGC ACC AGA CAT 1494 Ala His Asn Phe Met Gln Val Leu Gly Phe Arg Pro Ser Thr Arg His 470 475 480 CCC AAG TAT GAC AAG TTG TTC TTT GTC GGG GCT TCG ACT CAT CCC GGA 1542 Pro Lys Tyr Asp Lys Leu Phe Phe Val Gly Ala Ser Thr His Pro Gly 485 490 495 ACT GGG GTT CCC ATC GTC TTG GCT GGA GCC AAG TTA ACT GCC AAC CAA 1590 Thr Gly Val Pro Ile Val Leu Ala Gly Ala Lys Leu Thr Ala Asn Gln 500 505 510 515 GTT CTC GAA TCC TTT GAC CGA TCC CCA GCT CCA GAT CCC AAT ATG TCA 1638 Val Leu Glu Ser Phe Asp Arg Ser Pro Ala Pro Asp Pro Asn Met Ser 520 525 530 CTC TCC GTA CCA TAT GGA AAA CCT CTC AAA TCA AAT GGA ACG GGT ATC 1686 Leu Ser Val Pro Tyr Gly Lys Pro Leu Lys Ser Asn Gly Thr Gly Ile 535 540 545 GAT TCT CAG GTC CAG CTG AAG TTC ATG GAT TTG GAG AGA TGG GTA TAC 1734 Asp Ser Gln Val Gln Leu Lys Phe Met Asp Leu Glu Arg Trp Val Tyr 550 555 560 CTT TTG GTG TTG TTG ATT GGG GCC GTG ATC GCT CGA TCC GTT GGT GTT 1782 Leu Leu Val Leu Leu Ile Gly Ala Val Ile Ala Arg Ser Val Gly Val 565 570 575 CTT GCT TTC TGAAGCAAGA CAACGATCGT TTCTTAGAGT TTTTTTTAGT 1831 Leu Ala Phe 580 CTCTTCCTGT GTTCTCTCTA TATACATACT CTGCTCGTCT GTTCTCTTCT CGAGGGTTCC 1891 TCTTTACTTT GTGTCAGAGT CATACCCGGT CTCTCTCAAC GTCCGTTTGA GGGCTAGACA 1951 ATTGTTAGTC TCGAAATCTC CATCACCTCA AGTCTGATGT TCATCATCTT TTTTATTCGT 2011 TGCAATATAC ATGACTGTTA TGGACCGAAA AAAAAAAAAA AAAAAAA 2058 582 amino acids amino acid linear protein not provided 17 Met Gly Lys Glu Gln Asp Gln Asp Lys Pro Thr Ala Ile Ile Val Gly 1 5 10 15 Cys Gly Ile Gly Gly Ile Ala Thr Ala Ala Arg Leu Ala Lys Glu Gly 20 25 30 Phe Gln Val Thr Val Phe Glu Lys Asn Asp Tyr Ser Gly Gly Arg Cys 35 40 45 Ser Leu Ile Glu Arg Asp Gly Tyr Arg Phe Asp Gln Gly Pro Ser Leu 50 55 60 Leu Leu Leu Pro Asp Leu Phe Lys Gln Thr Phe Glu Asp Leu Gly Glu 65 70 75 80 Lys Met Glu Asp Trp Val Asp Leu Ile Lys Cys Glu Pro Asn Tyr Val 85 90 95 Cys His Phe His Asp Glu Glu Thr Phe Thr Phe Ser Thr Asp Met Ala 100 105 110 Leu Leu Lys Arg Glu Val Glu Arg Phe Glu Gly Lys Asp Gly Phe Asp 115 120 125 Arg Phe Leu Ser Phe Ile Gln Glu Ala His Arg His Tyr Glu Leu Ala 130 135 140 Val Val His Val Leu Gln Lys Asn Phe Pro Gly Phe Ala Ala Phe Leu 145 150 155 160 Arg Leu Gln Phe Ile Gly Gln Ile Leu Ala Leu His Pro Phe Glu Ser 165 170 175 Ile Trp Thr Arg Val Cys Arg Tyr Phe Lys Thr Asp Arg Leu Arg Arg 180 185 190 Val Phe Ser Phe Ala Val Met Tyr Met Gly Gln Ser Pro Tyr Ser Ala 195 200 205 Pro Gly Thr Tyr Ser Leu Leu Gln Tyr Thr Glu Leu Thr Glu Gly Ile 210 215 220 Trp Tyr Pro Arg Gly Gly Phe Trp Gln Val Pro Asn Thr Leu Leu Gln 225 230 235 240 Ile Val Lys Arg Asn Asn Pro Ser Ala Lys Phe Asn Phe Asn Ala Pro 245 250 255 Val Ser Gln Val Leu Leu Ser Pro Ala Lys Asp Arg Ala Thr Gly Val 260 265 270 Arg Leu Glu Ser Gly Glu Glu His His Ala Asp Val Val Ile Val Asn 275 280 285 Ala Asp Leu Val Tyr Ala Ser Glu His Leu Ile Pro Asp Asp Ala Arg 290 295 300 Asn Lys Ile Gly Gln Leu Gly Glu Val Lys Arg Ser Trp Trp Ala Asp 305 310 315 320 Leu Val Gly Gly Lys Lys Leu Lys Gly Ser Cys Ser Ser Leu Ser Phe 325 330 335 Tyr Trp Ser Met Asp Arg Ile Val Asp Gly Leu Gly Gly His Asn Ile 340 345 350 Phe Leu Ala Glu Asp Phe Lys Gly Ser Phe Asp Thr Ile Phe Glu Glu 355 360 365 Leu Gly Leu Pro Ala Asp Pro Ser Phe Tyr Val Asn Val Pro Ser Arg 370 375 380 Ile Asp Pro Ser Ala Ala Pro Glu Gly Lys Asp Ala Ile Val Ile Leu 385 390 395 400 Val Pro Cys Gly His Ile Asp Ala Ser Asn Pro Gln Asp Tyr Asn Lys 405 410 415 Leu Val Ala Arg Ala Arg Lys Phe Val Ile Gln Thr Leu Ser Ala Lys 420 425 430 Leu Gly Leu Pro Asp Phe Glu Lys Met Ile Val Ala Glu Lys Val His 435 440 445 Asp Ala Pro Ser Trp Glu Lys Glu Phe Asn Leu Lys Asp Gly Ser Ile 450 455 460 Leu Gly Leu Ala His Asn Phe Met Gln Val Leu Gly Phe Arg Pro Ser 465 470 475 480 Thr Arg His Pro Lys Tyr Asp Lys Leu Phe Phe Val Gly Ala Ser Thr 485 490 495 His Pro Gly Thr Gly Val Pro Ile Val Leu Ala Gly Ala Lys Leu Thr 500 505 510 Ala Asn Gln Val Leu Glu Ser Phe Asp Arg Ser Pro Ala Pro Asp Pro 515 520 525 Asn Met Ser Leu Ser Val Pro Tyr Gly Lys Pro Leu Lys Ser Asn Gly 530 535 540 Thr Gly Ile Asp Ser Gln Val Gln Leu Lys Phe Met Asp Leu Glu Arg 545 550 555 560 Trp Val Tyr Leu Leu Val Leu Leu Ile Gly Ala Val Ile Ala Arg Ser 565 570 575 Val Gly Val Leu Ala Phe 580 2470 base pairs nucleic acid double linear cDNA NO NO Phaffia rhodozyma CDS 177..2198 /product= “PRcrtY” 18 AACAAGAAGT GGACACAGAG AGATCTTTGC TGAAGAGTTG TATTCCAGAA AGGGAAAACA 60 AAGGAAAGAA GCGCCGAAGC ACATCACCAA CTTCAGCAAG CCGGTCCAGC CCGATCTCGG 120 ATAGACATCA TCTTACCCAA CTCGTATCAT CCCCAACAGA TAGAGTTTTT GTCGCA 176 ATG ACG GCT CTC GCA TAT TAC CAG ATC CAT CTG ATC TAT ACT CTC CCA 224 Met Thr Ala Leu Ala Tyr Tyr Gln Ile His Leu Ile Tyr Thr Leu Pro 1 5 10 15 ATT CTT GGT CTT CTC GGC CTG CTC ACT TCC CCG ATT TTG ACA AAA TTT 272 Ile Leu Gly Leu Leu Gly Leu Leu Thr Ser Pro Ile Leu Thr Lys Phe 20 25 30 GAC ATC TAC AAA ATA TCG ATC CTC GTA TTT ATT GCG TTT AGT GCA ACC 320 Asp Ile Tyr Lys Ile Ser Ile Leu Val Phe Ile Ala Phe Ser Ala Thr 35 40 45 ACA CCA TGG GAC TCA TGG ATC ATC AGA AAT GGC GCA TGG ACA TAT CCA 368 Thr Pro Trp Asp Ser Trp Ile Ile Arg Asn Gly Ala Trp Thr Tyr Pro 50 55 60 TCA GCG GAG AGT GGC CAA GGC GTG TTT GGA ACG TTT CTA GAT GTT CCA 416 Ser Ala Glu Ser Gly Gln Gly Val Phe Gly Thr Phe Leu Asp Val Pro 65 70 75 80 TAT GAA GAG TAC GCT TTC TTT GTC ATT CAA ACC GTA ATC ACC GGC TTG 464 Tyr Glu Glu Tyr Ala Phe Phe Val Ile Gln Thr Val Ile Thr Gly Leu 85 90 95 GTC TAC GTC TTG GCA ACT AGG CAC CTT CTC CCA TCT CTC GCG CTT CCC 512 Val Tyr Val Leu Ala Thr Arg His Leu Leu Pro Ser Leu Ala Leu Pro 100 105 110 AAG ACT AGA TCG TCC GCC CTT TCT CTC GCG CTC AAG GCG CTC ATC CCT 560 Lys Thr Arg Ser Ser Ala Leu Ser Leu Ala Leu Lys Ala Leu Ile Pro 115 120 125 CTG CCC ATT ATC TAC CTA TTT ACC GCT CAC CCC AGC CCA TCG CCC GAC 608 Leu Pro Ile Ile Tyr Leu Phe Thr Ala His Pro Ser Pro Ser Pro Asp 130 135 140 CCG CTC GTG ACA GAT CAC TAC TTC TAC ATG CGG GCA CTC TCC TTA CTC 656 Pro Leu Val Thr Asp His Tyr Phe Tyr Met Arg Ala Leu Ser Leu Leu 145 150 155 160 ATC ACC CCA CCT ACC ATG CTC TTG GCA GCA TTA TCA GGC GAA TAT GCT 704 Ile Thr Pro Pro Thr Met Leu Leu Ala Ala Leu Ser Gly Glu Tyr Ala 165 170 175 TTC GAT TGG AAA AGT GGC CGA GCA AAG TCA ACT ATT GCA GCA ATC ATG 752 Phe Asp Trp Lys Ser Gly Arg Ala Lys Ser Thr Ile Ala Ala Ile Met 180 185 190 ATC CCG ACG GTG TAT CTG ATT TGG GTA GAT TAT GTT GCT GTC GGT CAA 800 Ile Pro Thr Val Tyr Leu Ile Trp Val Asp Tyr Val Ala Val Gly Gln 195 200 205 GAC TCT TGG TCG ATC AAC GAT GAG AAG ATT GTA GGG TGG AGG CTT GGA 848 Asp Ser Trp Ser Ile Asn Asp Glu Lys Ile Val Gly Trp Arg Leu Gly 210 215 220 GGT GTA CTA CCC ATT GAG GAA GCT ATG TTC TTC TTA CTG ACG AAT CTA 896 Gly Val Leu Pro Ile Glu Glu Ala Met Phe Phe Leu Leu Thr Asn Leu 225 230 235 240 ATG ATT GTT CTG GGT CTG TCT GCC TGC GAT CAT ACT CAG GCC CTA TAC 944 Met Ile Val Leu Gly Leu Ser Ala Cys Asp His Thr Gln Ala Leu Tyr 245 250 255 CTG CTA CAC GGT CGA ACT ATT TAT GGC AAC AAA AAG ATG CCA TCT TCA 992 Leu Leu His Gly Arg Thr Ile Tyr Gly Asn Lys Lys Met Pro Ser Ser 260 265 270 TTT CCC CTC ATT ACA CCG CCT GTG CTC TCC CTG TTT TTT AGC AGC CGA 1040 Phe Pro Leu Ile Thr Pro Pro Val Leu Ser Leu Phe Phe Ser Ser Arg 275 280 285 CCA TAC TCT TCT CAG CCA AAA CGT GAC TTG GAA CTG GCA GTC AAG TTG 1088 Pro Tyr Ser Ser Gln Pro Lys Arg Asp Leu Glu Leu Ala Val Lys Leu 290 295 300 TTG GAG AAA AAG AGC CGG AGC TTT TTT GTT GCC TCG GCT GGA TTT CCT 1136 Leu Glu Lys Lys Ser Arg Ser Phe Phe Val Ala Ser Ala Gly Phe Pro 305 310 315 320 AGC GAA GTT AGG GAG AGG CTG GTT GGA CTA TAC GCA TTC TGC CGG GTG 1184 Ser Glu Val Arg Glu Arg Leu Val Gly Leu Tyr Ala Phe Cys Arg Val 325 330 335 ACT GAT GAT CTT ATC GAC TCT CCT GAA GTA TCT TCC AAC CCG CAT GCC 1232 Thr Asp Asp Leu Ile Asp Ser Pro Glu Val Ser Ser Asn Pro His Ala 340 345 350 ACA ATT GAC ATG GTC TCC GAT TTT CTT ACC CTA CTA TTT GGG CCC CCG 1280 Thr Ile Asp Met Val Ser Asp Phe Leu Thr Leu Leu Phe Gly Pro Pro 355 360 365 CTA CAC CCT TCG CAA CCT GAC AAG ATC CTT TCT TCG CCT TTA CTT CCT 1328 Leu His Pro Ser Gln Pro Asp Lys Ile Leu Ser Ser Pro Leu Leu Pro 370 375 380 CCT TCG CAC CCT TCC CGA CCC ACG GGA ATG TAT CCC CTC CCG CCT CCT 1376 Pro Ser His Pro Ser Arg Pro Thr Gly Met Tyr Pro Leu Pro Pro Pro 385 390 395 400 CCT TCG CTC TCG CCT GCC GAG CTC GTT CAA TTC CTT ACC GAA AGG GTT 1424 Pro Ser Leu Ser Pro Ala Glu Leu Val Gln Phe Leu Thr Glu Arg Val 405 410 415 CCC GTT CAA TAC CAT TTC GCC TTC AGG TTG CTC GCT AAG TTG CAA GGG 1472 Pro Val Gln Tyr His Phe Ala Phe Arg Leu Leu Ala Lys Leu Gln Gly 420 425 430 CTG ATC CCT CGA TAC CCA CTC GAC GAA CTC CTT AGA GGA TAC ACC ACT 1520 Leu Ile Pro Arg Tyr Pro Leu Asp Glu Leu Leu Arg Gly Tyr Thr Thr 435 440 445 GAT CTT ATC TTT CCC TTA TCG ACA GAG GCA GTC CAG GCT CGG AAG ACG 1568 Asp Leu Ile Phe Pro Leu Ser Thr Glu Ala Val Gln Ala Arg Lys Thr 450 455 460 CCT ATC GAG ACC ACA GCT GAC TTG CTG GAC TAT GGT CTA TGT GTA GCA 1616 Pro Ile Glu Thr Thr Ala Asp Leu Leu Asp Tyr Gly Leu Cys Val Ala 465 470 475 480 GGC TCA GTC GCC GAG CTA TTG GTC TAT GTC TCT TGG GCA AGT GCA CCA 1664 Gly Ser Val Ala Glu Leu Leu Val Tyr Val Ser Trp Ala Ser Ala Pro 485 490 495 AGT CAG GTC CCT GCC ACC ATA GAA GAA AGA GAA GCT GTG TTA GTG GCA 1712 Ser Gln Val Pro Ala Thr Ile Glu Glu Arg Glu Ala Val Leu Val Ala 500 505 510 AGC CGA GAG ATG GGA ACT GCC CTT CAG TTG GTG AAC ATT GCT AGG GAC 1760 Ser Arg Glu Met Gly Thr Ala Leu Gln Leu Val Asn Ile Ala Arg Asp 515 520 525 ATT AAA GGG GAC GCA ACA GAA GGG AGA TTT TAC CTA CCA CTC TCA TTC 1808 Ile Lys Gly Asp Ala Thr Glu Gly Arg Phe Tyr Leu Pro Leu Ser Phe 530 535 540 TTT GGT CTT CGG GAT GAA TCA AAG CTT GCG ATC CCG ACT GAT TGG ACG 1856 Phe Gly Leu Arg Asp Glu Ser Lys Leu Ala Ile Pro Thr Asp Trp Thr 545 550 555 560 GAA CCT CGG CCT CAA GAT TTC GAC AAA CTC CTC AGT CTA TCT CCT TCG 1904 Glu Pro Arg Pro Gln Asp Phe Asp Lys Leu Leu Ser Leu Ser Pro Ser 565 570 575 TCC ACA TTA CCA TCT TCA AAC GCC TCA GAA AGC TTC CGG TTC GAA TGG 1952 Ser Thr Leu Pro Ser Ser Asn Ala Ser Glu Ser Phe Arg Phe Glu Trp 580 585 590 AAG ACG TAC TCG CTT CCA TTA GTC GCC TAC GCA GAG GAT CTT GCC AAA 2000 Lys Thr Tyr Ser Leu Pro Leu Val Ala Tyr Ala Glu Asp Leu Ala Lys 595 600 605 CAT TCT TAT AAG GGA ATT GAC CGA CTT CCT ACC GAG GTT CAA GCG GGA 2048 His Ser Tyr Lys Gly Ile Asp Arg Leu Pro Thr Glu Val Gln Ala Gly 610 615 620 ATG CGA GCG GCT TGC GCG AGC TAC CTA CTG ATC GGC CGA GAG ATC AAA 2096 Met Arg Ala Ala Cys Ala Ser Tyr Leu Leu Ile Gly Arg Glu Ile Lys 625 630 635 640 GTC GTT TGG AAA GGA GAC GTC GGA GAG AGA AGG ACA GTT GCC GGA TGG 2144 Val Val Trp Lys Gly Asp Val Gly Glu Arg Arg Thr Val Ala Gly Trp 645 650 655 AGG AGA GTA CGG AAA GTC TTG AGT GTG GTC ATG AGC GGA TGG GAA GGG 2192 Arg Arg Val Arg Lys Val Leu Ser Val Val Met Ser Gly Trp Glu Gly 660 665 670 CAG TAAGACAGCG GAAGAATACC GACAGACAAT GATGAGTGAG AATAAAATCA 2245 Gln TCCTCAATCT TCTTTCTCTA GGTGCTCTTT TTTGTTTTCT ATTATGACCA ACTCTAAAGG 2305 AACTGGCCTT GCAGATATTT CTCTTCCCCC CATCTTCCTC CTTTCCATCG TTTGTTCTTT 2365 CCATTTTTGT CGGTTTACTA TGTCAATTCT TTTTCTTGCT TTTTCTTATC AATCTAGACA 2425 ATTCTATAGA TGTTTAGAAT TTATACAAAA AAAAAAAAAA AAAAA 2470 673 amino acids amino acid linear protein not provided 19 Met Thr Ala Leu Ala Tyr Tyr Gln Ile His Leu Ile Tyr Thr Leu Pro 1 5 10 15 Ile Leu Gly Leu Leu Gly Leu Leu Thr Ser Pro Ile Leu Thr Lys Phe 20 25 30 Asp Ile Tyr Lys Ile Ser Ile Leu Val Phe Ile Ala Phe Ser Ala Thr 35 40 45 Thr Pro Trp Asp Ser Trp Ile Ile Arg Asn Gly Ala Trp Thr Tyr Pro 50 55 60 Ser Ala Glu Ser Gly Gln Gly Val Phe Gly Thr Phe Leu Asp Val Pro 65 70 75 80 Tyr Glu Glu Tyr Ala Phe Phe Val Ile Gln Thr Val Ile Thr Gly Leu 85 90 95 Val Tyr Val Leu Ala Thr Arg His Leu Leu Pro Ser Leu Ala Leu Pro 100 105 110 Lys Thr Arg Ser Ser Ala Leu Ser Leu Ala Leu Lys Ala Leu Ile Pro 115 120 125 Leu Pro Ile Ile Tyr Leu Phe Thr Ala His Pro Ser Pro Ser Pro Asp 130 135 140 Pro Leu Val Thr Asp His Tyr Phe Tyr Met Arg Ala Leu Ser Leu Leu 145 150 155 160 Ile Thr Pro Pro Thr Met Leu Leu Ala Ala Leu Ser Gly Glu Tyr Ala 165 170 175 Phe Asp Trp Lys Ser Gly Arg Ala Lys Ser Thr Ile Ala Ala Ile Met 180 185 190 Ile Pro Thr Val Tyr Leu Ile Trp Val Asp Tyr Val Ala Val Gly Gln 195 200 205 Asp Ser Trp Ser Ile Asn Asp Glu Lys Ile Val Gly Trp Arg Leu Gly 210 215 220 Gly Val Leu Pro Ile Glu Glu Ala Met Phe Phe Leu Leu Thr Asn Leu 225 230 235 240 Met Ile Val Leu Gly Leu Ser Ala Cys Asp His Thr Gln Ala Leu Tyr 245 250 255 Leu Leu His Gly Arg Thr Ile Tyr Gly Asn Lys Lys Met Pro Ser Ser 260 265 270 Phe Pro Leu Ile Thr Pro Pro Val Leu Ser Leu Phe Phe Ser Ser Arg 275 280 285 Pro Tyr Ser Ser Gln Pro Lys Arg Asp Leu Glu Leu Ala Val Lys Leu 290 295 300 Leu Glu Lys Lys Ser Arg Ser Phe Phe Val Ala Ser Ala Gly Phe Pro 305 310 315 320 Ser Glu Val Arg Glu Arg Leu Val Gly Leu Tyr Ala Phe Cys Arg Val 325 330 335 Thr Asp Asp Leu Ile Asp Ser Pro Glu Val Ser Ser Asn Pro His Ala 340 345 350 Thr Ile Asp Met Val Ser Asp Phe Leu Thr Leu Leu Phe Gly Pro Pro 355 360 365 Leu His Pro Ser Gln Pro Asp Lys Ile Leu Ser Ser Pro Leu Leu Pro 370 375 380 Pro Ser His Pro Ser Arg Pro Thr Gly Met Tyr Pro Leu Pro Pro Pro 385 390 395 400 Pro Ser Leu Ser Pro Ala Glu Leu Val Gln Phe Leu Thr Glu Arg Val 405 410 415 Pro Val Gln Tyr His Phe Ala Phe Arg Leu Leu Ala Lys Leu Gln Gly 420 425 430 Leu Ile Pro Arg Tyr Pro Leu Asp Glu Leu Leu Arg Gly Tyr Thr Thr 435 440 445 Asp Leu Ile Phe Pro Leu Ser Thr Glu Ala Val Gln Ala Arg Lys Thr 450 455 460 Pro Ile Glu Thr Thr Ala Asp Leu Leu Asp Tyr Gly Leu Cys Val Ala 465 470 475 480 Gly Ser Val Ala Glu Leu Leu Val Tyr Val Ser Trp Ala Ser Ala Pro 485 490 495 Ser Gln Val Pro Ala Thr Ile Glu Glu Arg Glu Ala Val Leu Val Ala 500 505 510 Ser Arg Glu Met Gly Thr Ala Leu Gln Leu Val Asn Ile Ala Arg Asp 515 520 525 Ile Lys Gly Asp Ala Thr Glu Gly Arg Phe Tyr Leu Pro Leu Ser Phe 530 535 540 Phe Gly Leu Arg Asp Glu Ser Lys Leu Ala Ile Pro Thr Asp Trp Thr 545 550 555 560 Glu Pro Arg Pro Gln Asp Phe Asp Lys Leu Leu Ser Leu Ser Pro Ser 565 570 575 Ser Thr Leu Pro Ser Ser Asn Ala Ser Glu Ser Phe Arg Phe Glu Trp 580 585 590 Lys Thr Tyr Ser Leu Pro Leu Val Ala Tyr Ala Glu Asp Leu Ala Lys 595 600 605 His Ser Tyr Lys Gly Ile Asp Arg Leu Pro Thr Glu Val Gln Ala Gly 610 615 620 Met Arg Ala Ala Cys Ala Ser Tyr Leu Leu Ile Gly Arg Glu Ile Lys 625 630 635 640 Val Val Trp Lys Gly Asp Val Gly Glu Arg Arg Thr Val Ala Gly Trp 645 650 655 Arg Arg Val Arg Lys Val Leu Ser Val Val Met Ser Gly Trp Glu Gly 660 665 670 Gln 1165 base pairs nucleic acid double linear cDNA NO NO Phaffia rhodozyma CDS 141..896 /product= “PRidi” 20 CTTCTCTTTC CTCGACCTCT TCGGCAGGCC GTTGAAGACT CGTTTACTCA TACCCCACAT 60 CTCGCATATA TCACTTTCCT CCTTCCAGAA CAAGTTCTGA GTCAACCGAA AAGAAAGAAG 120 GCAGAAGAAA TATATTCTAG ATG TCC ATG CCC AAC ATT GTT CCC CCC GCC 170 Met Ser Met Pro Asn Ile Val Pro Pro Ala 1 5 10 GAG GTC CGA ACC GAA GGA CTC AGT TTA GAA GAG TAC GAT GAG GAG CAG 218 Glu Val Arg Thr Glu Gly Leu Ser Leu Glu Glu Tyr Asp Glu Glu Gln 15 20 25 GTC AGG CTG ATG GAG GAG CGA TGT ATT CTT GTT AAC CCG GAC GAT GTG 266 Val Arg Leu Met Glu Glu Arg Cys Ile Leu Val Asn Pro Asp Asp Val 30 35 40 GCC TAT GGA GAG GCT TCG AAA AAG ACC TGC CAC TTG ATG TCC AAC ATC 314 Ala Tyr Gly Glu Ala Ser Lys Lys Thr Cys His Leu Met Ser Asn Ile 45 50 55 AAC GCG CCC AAG GAC CTC CTC CAC CGA GCA TTC TCC GTG TTT CTC TTC 362 Asn Ala Pro Lys Asp Leu Leu His Arg Ala Phe Ser Val Phe Leu Phe 60 65 70 CGC CCA TCG GAC GGA GCA CTC CTG CTT CAG CGA AGA GCG GAC GAG AAG 410 Arg Pro Ser Asp Gly Ala Leu Leu Leu Gln Arg Arg Ala Asp Glu Lys 75 80 85 90 ATT ACG TTC CCT GGA ATG TGG ACC AAC ACG TGT TGC AGT CAT CCT TTG 458 Ile Thr Phe Pro Gly Met Trp Thr Asn Thr Cys Cys Ser His Pro Leu 95 100 105 AGC ATC AAG GGC GAG GTT GAA GAG GAG AAC CAG ATC GGT GTT CGA CGA 506 Ser Ile Lys Gly Glu Val Glu Glu Glu Asn Gln Ile Gly Val Arg Arg 110 115 120 GCT GCG TCC CGA AAG TTG GAG CAC GAG CTT GGC GTG CCT ACA TCG TCG 554 Ala Ala Ser Arg Lys Leu Glu His Glu Leu Gly Val Pro Thr Ser Ser 125 130 135 ACT CCG CCC GAC TCG TTC ACC TAC CTC ACT AGG ATA CAT TAC CTC GCT 602 Thr Pro Pro Asp Ser Phe Thr Tyr Leu Thr Arg Ile His Tyr Leu Ala 140 145 150 CCG AGT GAC GGA CTC TGG GGA GAA CAC GAG ATC GAC TAC ATT CTC TTC 650 Pro Ser Asp Gly Leu Trp Gly Glu His Glu Ile Asp Tyr Ile Leu Phe 155 160 165 170 TCA ACC ACA CCT ACA GAA CAC ACT GGA AAC CCT AAC GAA GTC TCT GAC 698 Ser Thr Thr Pro Thr Glu His Thr Gly Asn Pro Asn Glu Val Ser Asp 175 180 185 ACT CGA TAT GTC ACC AAG CCC GAG CTC CAG GCG ATG TTT GAG GAC GAG 746 Thr Arg Tyr Val Thr Lys Pro Glu Leu Gln Ala Met Phe Glu Asp Glu 190 195 200 TCT AAC TCA TTT ACC CCT TGG TTC AAA TTG ATT GCC CGA GAC TTC CTG 794 Ser Asn Ser Phe Thr Pro Trp Phe Lys Leu Ile Ala Arg Asp Phe Leu 205 210 215 TTT GGC TGG TGG GAT CAA CTT CTC GCC AGA CGA AAT GAA AAG GGT GAG 842 Phe Gly Trp Trp Asp Gln Leu Leu Ala Arg Arg Asn Glu Lys Gly Glu 220 225 230 GTC GAT GCC AAA TCG TTG GAG GAT CTC TCG GAC AAC AAA GTC TGG AAG 890 Val Asp Ala Lys Ser Leu Glu Asp Leu Ser Asp Asn Lys Val Trp Lys 235 240 245 250 ATG TAGTCGACCC TTCTTTCTGT ACAGTCATCT CAGTTCGCCT GTTGGTTGCT 943 Met TGCTTCTTGC TCTTCTTTCT ATATATCTTT TTTCTTGCCT GGGTAGACTT GATCTTTCTA 1003 CATAGCATAC GCATACATAC ATAAACTCTA TTTCTTGTTC TTTATCTCTC TTCTAAGGGA 1063 ATCTTCAAGA TCAATTTCTT TTTGGGCTAC AACATTTCAG ATCAATATTG CTTTTCAGAC 1123 TACAAAAAAA AAAAAAAAAA ACTCGAGGGG GGGCCCGGTA CC 1165 251 amino acids amino acid linear protein not provided 21 Met Ser Met Pro Asn Ile Val Pro Pro Ala Glu Val Arg Thr Glu Gly 1 5 10 15 Leu Ser Leu Glu Glu Tyr Asp Glu Glu Gln Val Arg Leu Met Glu Glu 20 25 30 Arg Cys Ile Leu Val Asn Pro Asp Asp Val Ala Tyr Gly Glu Ala Ser 35 40 45 Lys Lys Thr Cys His Leu Met Ser Asn Ile Asn Ala Pro Lys Asp Leu 50 55 60 Leu His Arg Ala Phe Ser Val Phe Leu Phe Arg Pro Ser Asp Gly Ala 65 70 75 80 Leu Leu Leu Gln Arg Arg Ala Asp Glu Lys Ile Thr Phe Pro Gly Met 85 90 95 Trp Thr Asn Thr Cys Cys Ser His Pro Leu Ser Ile Lys Gly Glu Val 100 105 110 Glu Glu Glu Asn Gln Ile Gly Val Arg Arg Ala Ala Ser Arg Lys Leu 115 120 125 Glu His Glu Leu Gly Val Pro Thr Ser Ser Thr Pro Pro Asp Ser Phe 130 135 140 Thr Tyr Leu Thr Arg Ile His Tyr Leu Ala Pro Ser Asp Gly Leu Trp 145 150 155 160 Gly Glu His Glu Ile Asp Tyr Ile Leu Phe Ser Thr Thr Pro Thr Glu 165 170 175 His Thr Gly Asn Pro Asn Glu Val Ser Asp Thr Arg Tyr Val Thr Lys 180 185 190 Pro Glu Leu Gln Ala Met Phe Glu Asp Glu Ser Asn Ser Phe Thr Pro 195 200 205 Trp Phe Lys Leu Ile Ala Arg Asp Phe Leu Phe Gly Trp Trp Asp Gln 210 215 220 Leu Leu Ala Arg Arg Asn Glu Lys Gly Glu Val Asp Ala Lys Ser Leu 225 230 235 240 Glu Asp Leu Ser Asp Asn Lys Val Trp Lys Met 245 250 3550 base pairs nucleic acid double linear DNA (genomic) NO NO Phaffia rhodozyma CBS 6938 exon 941..966 intron 967..1077 exon 1078..1284 intron 1285..1364 exon 1365..1877 intron 1878..1959 exon 1960..2202 intron 2203..2292 exon 2293..3325 CDS join(941..966, 1078..1284, 1365..1877, 1960..2202, 2293..3325) /product= “PRGcrtB GB” 22 GGAATTCCAG TTTTGCCTTT GACGAGAAAG GACACTGGGT TGGAAAGAGA AGATGGTACG 60 TTCTTCTCCA CCTTGAATGT GTTGCTTACT AGACATGTTT GACACGCTAA TGCATTTCTT 120 TCCACTTTGA CTTTTGAACT ATGGTGGTTG GGCGATCCCC AAAATCATTA GCTTCTACTT 180 CAGCTCATTA CCTCGATCTC ATCTTACTAC CAGGTGTTGC ATTCTCACCT ACGGCCTCTT 240 CTTTGTTCTC TCGACTGGGC CATGGAAAAG GATATTACGA TAAATACATC ACTCAGTATC 300 GGTCGATCTG TGCAGGCAAG AATCGACCCG TCCGAAGCTG AGTACGCGTC TTCTCTTTTC 360 TCGATACCCA ACGGACGCTA TTTTGTGACA GAAGGATGAG ACTATCCAAC AGCTCAAACA 420 AACTAACGCT CTTGATTAAT CACCCGCTCA ACTTATTGCT CAACTCAGTT GGACTGGCGC 480 TGAAAGAACA GTTCTTAGAC AAAAACATGG TCCCTATAGG AGAATGGGAT GCGAATCTGG 540 ATGAAGTGTT GGTTGGAGAT CACGTGAGGA CATTATCCGA GGACAATTAA CTACTTAAGA 600 TATATACATG ATTTATGTCG ATCGGCATCC AGCCGGGGAT TGATCGGCTG ATGGCCGGAA 660 ATGTGATGAT GGTCGAAACT CGATCTCTCT TTTTTTGTTC ATCTTCTCAT CCCTCTTCTC 720 TCTTTCTACT GACATCCATC TCCAACTGTC TAGATCAGTT CGGAAACAAG AAGTGGACAC 780 AGAGAGATCT TTGCTGAAGA GTTGTATTCC AGAAAGGGAA AACAAAGGAA AGAAGCGCCG 840 AAGCACATCA CCAACTTCAG CAAGCCGGTC CAGCCCGATC TCGGATAGAC ATCATCTTAC 900 CCAACTCGTA TCATCCCCAA CAGATAGAGT TTTTGTCGCA ATG ACG GCT CTC GCA 955 Met Thr Ala Leu Ala 1 5 TAT TAC CAG AT GTTTGTCTCC ATACCTCTTC TTCGTTTTGC ACACCACTCA 1006 Tyr Tyr Gln Ile TGTGTGCATA TGTGTGTGCG TCCTTCCAAA TCTTTCAATG ACTAACATCT TTACCGTGCT 1066 CTTCTTCTTA G C CAT CTG ATC TAT ACT CTC CCA ATT CTT GGT CTT CTC 1114 His Leu Ile Tyr Thr Leu Pro Ile Leu Gly Leu Leu 10 15 20 GGC CTG CTC ACT TCC CCG ATT TTG ACA AAA TTT GAC ATC TAC AAA ATA 1162 Gly Leu Leu Thr Ser Pro Ile Leu Thr Lys Phe Asp Ile Tyr Lys Ile 25 30 35 TCG ATC CTC GTA TTT ATT GCG TTT AGT GCA ACC ACA CCA TGG GAC TCA 1210 Ser Ile Leu Val Phe Ile Ala Phe Ser Ala Thr Thr Pro Trp Asp Ser 40 45 50 TGG ATC ATC AGA AAT GGC GCA TGG ACA TAT CCA TCA GCG GAG AGT GGC 1258 Trp Ile Ile Arg Asn Gly Ala Trp Thr Tyr Pro Ser Ala Glu Ser Gly 55 60 65 CAA GGC GTG TTT GGA ACG TTT CTA GA GTTAGTCGAC CGTTAATACT 1304 Gln Gly Val Phe Gly Thr Phe Leu Asp 70 75 CTTAGCCGCG CGTCGTTTCC GCGATTACAT TTAACATCTG AATTTATCCC TGATCAACAG 1364 T GTT CCA TAT GAA GAG TAC GCT TTC TTT GTC ATT CAA ACC GTA ATC 1410 Val Pro Tyr Glu Glu Tyr Ala Phe Phe Val Ile Gln Thr Val Ile 80 85 90 ACC GGC TTG GTC TAC GTC TTG GCA ACT AGG CAC CTT CTC CCA TCT CTC 1458 Thr Gly Leu Val Tyr Val Leu Ala Thr Arg His Leu Leu Pro Ser Leu 95 100 105 GCG CTT CCC AAG ACT AGA TCG TCC GCC CTT TCT CTC GCG CTC AAG GCG 1506 Ala Leu Pro Lys Thr Arg Ser Ser Ala Leu Ser Leu Ala Leu Lys Ala 110 115 120 125 CTC ATC CCT CTG CCC ATT ATC TAC CTA TTT ACC GCT CAC CCC AGC CCA 1554 Leu Ile Pro Leu Pro Ile Ile Tyr Leu Phe Thr Ala His Pro Ser Pro 130 135 140 TCG CCC GAC CCG CTC GTG ACA GAT CAC TAC TTC TAC ATG CGG GCA CTC 1602 Ser Pro Asp Pro Leu Val Thr Asp His Tyr Phe Tyr Met Arg Ala Leu 145 150 155 TCC TTA CTC ATC ACC CCA CCT ACC ATG CTC TTG GCA GCA TTA TCA GGC 1650 Ser Leu Leu Ile Thr Pro Pro Thr Met Leu Leu Ala Ala Leu Ser Gly 160 165 170 GAA TAT GCT TTC GAT TGG AAA AGT GGC CGA GCA AAG TCA ACT ATT GCA 1698 Glu Tyr Ala Phe Asp Trp Lys Ser Gly Arg Ala Lys Ser Thr Ile Ala 175 180 185 GCA ATC ATG ATC CCG ACG GTG TAT CTG ATT TGG GTA GAT TAT GTT GCT 1746 Ala Ile Met Ile Pro Thr Val Tyr Leu Ile Trp Val Asp Tyr Val Ala 190 195 200 205 GTC GGT CAA GAC TCT TGG TCG ATC AAC GAT GAG AAG ATT GTA GGG TGG 1794 Val Gly Gln Asp Ser Trp Ser Ile Asn Asp Glu Lys Ile Val Gly Trp 210 215 220 AGG CTT GGA GGT GTA CTA CCC ATT GAG GAA GCT ATG TTC TTC TTA CTG 1842 Arg Leu Gly Gly Val Leu Pro Ile Glu Glu Ala Met Phe Phe Leu Leu 225 230 235 ACG AAT CTA ATG ATT GTT CTG GGT CTG TCT GCC TG GTAAGTTGAT 1887 Thr Asn Leu Met Ile Val Leu Gly Leu Ser Ala Cys 240 245 CTCATCCTCT CTTCCTTTGG TGAAAAAAGC TGTTTGGCTG ATTGCTGCGA ACTCACCCAT 1947 CGGAATCTGT AG C GAT CAT ACT CAG GCC CTA TAC CTG CTA CAC GGT CGA 1996 Asp His Thr Gln Ala Leu Tyr Leu Leu His Gly Arg 250 255 260 ACT ATT TAT GGC AAC AAA AAG ATG CCA TCT TCA TTT CCC CTC ATT ACA 2044 Thr Ile Tyr Gly Asn Lys Lys Met Pro Ser Ser Phe Pro Leu Ile Thr 265 270 275 CCG CCT GTG CTC TCC CTG TTT TTT AGC AGC CGA CCA TAC TCT TCT CAG 2092 Pro Pro Val Leu Ser Leu Phe Phe Ser Ser Arg Pro Tyr Ser Ser Gln 280 285 290 CCA AAA CGT GAC TTG GAA CTG GCA GTC AAG TTG TTG GAG AAA AAG AGC 2140 Pro Lys Arg Asp Leu Glu Leu Ala Val Lys Leu Leu Glu Lys Lys Ser 295 300 305 CGG AGC TTT TTT GTT GCC TCG GCT GGA TTT CCT AGC GAA GTT AGG GAG 2188 Arg Ser Phe Phe Val Ala Ser Ala Gly Phe Pro Ser Glu Val Arg Glu 310 315 320 325 AGG CTG GTT GGA CT GTGAGCACGC ATTCTTTAGG TTTGTTCGGT CTTTCACCTT 2242 Arg Leu Val Gly Leu 330 CATGTGCATT CGCTGATCAG TTTTCTTGGT GATCCGGGAC CTGCATACAG A TAC GCA 2299 Tyr Ala TTC TGC CGG GTG ACT GAT GAT CTT ATC GAC TCT CCT GAA GTA TCT TCC 2347 Phe Cys Arg Val Thr Asp Asp Leu Ile Asp Ser Pro Glu Val Ser Ser 335 340 345 AAC CCG CAT GCC ACA ATT GAC ATG GTC TCC GAT TTT CTT ACC CTA CTA 2395 Asn Pro His Ala Thr Ile Asp Met Val Ser Asp Phe Leu Thr Leu Leu 350 355 360 TTT GGG CCC CCG CTA CAC CCT TCG CAA CCT GAC AAG ATC CTT TCT TCG 2443 Phe Gly Pro Pro Leu His Pro Ser Gln Pro Asp Lys Ile Leu Ser Ser 365 370 375 380 CCT TTA CTT CCT CCT TCG CAC CCT TCC CGA CCC ACG GGA ATG TAT CCC 2491 Pro Leu Leu Pro Pro Ser His Pro Ser Arg Pro Thr Gly Met Tyr Pro 385 390 395 CTC CCG CCT CCT CCT TCG CTC TCG CCT GCC GAG CTC GTT CAA TTC CTT 2539 Leu Pro Pro Pro Pro Ser Leu Ser Pro Ala Glu Leu Val Gln Phe Leu 400 405 410 ACC GAA AGG GTT CCC GTT CAA TAC CAT TTC GCC TTC AGG TTG CTC GCT 2587 Thr Glu Arg Val Pro Val Gln Tyr His Phe Ala Phe Arg Leu Leu Ala 415 420 425 AAG TTG CAA GGG CTG ATC CCT CGA TAC CCA CTC GAC GAA CTC CTT AGA 2635 Lys Leu Gln Gly Leu Ile Pro Arg Tyr Pro Leu Asp Glu Leu Leu Arg 430 435 440 GGA TAC ACC ACT GAT CTT ATC TTT CCC TTA TCG ACA GAG GCA GTC CAG 2683 Gly Tyr Thr Thr Asp Leu Ile Phe Pro Leu Ser Thr Glu Ala Val Gln 445 450 455 460 GCT CGG AAG ACG CCT ATC GAG ACC ACA GCT GAC TTG CTG GAC TAT GGT 2731 Ala Arg Lys Thr Pro Ile Glu Thr Thr Ala Asp Leu Leu Asp Tyr Gly 465 470 475 CTA TGT GTA GCA GGC TCA GTC GCC GAG CTA TTG GTC TAT GTC TCT TGG 2779 Leu Cys Val Ala Gly Ser Val Ala Glu Leu Leu Val Tyr Val Ser Trp 480 485 490 GCA AGT GCA CCA AGT CAG GTC CCT GCC ACC ATA GAA GAA AGA GAA GCT 2827 Ala Ser Ala Pro Ser Gln Val Pro Ala Thr Ile Glu Glu Arg Glu Ala 495 500 505 GTG TTA GTG GCA AGC CGA GAG ATG GGA ACT GCC CTT CAG TTG GTG AAC 2875 Val Leu Val Ala Ser Arg Glu Met Gly Thr Ala Leu Gln Leu Val Asn 510 515 520 ATT GCT AGG GAC ATT AAA GGG GAC GCA ACA GAA GGG AGA TTT TAC CTA 2923 Ile Ala Arg Asp Ile Lys Gly Asp Ala Thr Glu Gly Arg Phe Tyr Leu 525 530 535 540 CCA CTC TCA TTC TTT GGT CTT CGG GAT GAA TCA AAG CTT GCG ATC CCG 2971 Pro Leu Ser Phe Phe Gly Leu Arg Asp Glu Ser Lys Leu Ala Ile Pro 545 550 555 ACT GAT TGG ACG GAA CCT CGG CCT CAA GAT TTC GAC AAA CTC CTC AGT 3019 Thr Asp Trp Thr Glu Pro Arg Pro Gln Asp Phe Asp Lys Leu Leu Ser 560 565 570 CTA TCT CCT TCG TCC ACA TTA CCA TCT TCA AAC GCC TCA GAA AGC TTC 3067 Leu Ser Pro Ser Ser Thr Leu Pro Ser Ser Asn Ala Ser Glu Ser Phe 575 580 585 CGG TTC GAA TGG AAG ACG TAC TCG CTT CCA TTA GTC GCC TAC GCA GAG 3115 Arg Phe Glu Trp Lys Thr Tyr Ser Leu Pro Leu Val Ala Tyr Ala Glu 590 595 600 GAT CTT GCC AAA CAT TCT TAT AAG GGA ATT GAC CGA CTT CCT ACC GAG 3163 Asp Leu Ala Lys His Ser Tyr Lys Gly Ile Asp Arg Leu Pro Thr Glu 605 610 615 620 GTT CAA GCG GGA ATG CGA GCG GCT TGC GCG AGC TAC CTA CTG ATC GGC 3211 Val Gln Ala Gly Met Arg Ala Ala Cys Ala Ser Tyr Leu Leu Ile Gly 625 630 635 CGA GAG ATC AAA GTC GTT TGG AAA GGA GAC GTC GGA GAG AGA AGG ACA 3259 Arg Glu Ile Lys Val Val Trp Lys Gly Asp Val Gly Glu Arg Arg Thr 640 645 650 GTT GCC GGA TGG AGG AGA GTA CGG AAA GTC TTG AGT GTG GTC ATG AGC 3307 Val Ala Gly Trp Arg Arg Val Arg Lys Val Leu Ser Val Val Met Ser 655 660 665 GGA TGG GAA GGG CAG TAAGACAGCG GAAGAATACC GACAGACAAT GATGAGTGAG 3362 Gly Trp Glu Gly Gln 670 AATAAAATCA TCCTCAATCT TCTTTCTCTA GGTGCTCTTT TTTGTTTTCT ATTATGACCA 3422 ACTCTAAAGG AACTGGCCTT GCAGATATTT CTCTTCCCCC CATCTTCCTC CTTTCCATCG 3482 TTTGTTCTTT CCATTTTTGT CGGTTTACTA TGTCAATTCT TTTTCTTGCT TTTTCTTATC 3542 AATCTAGA 3550 673 amino acids amino acid linear protein not provided 23 Met Thr Ala Leu Ala Tyr Tyr Gln Ile His Leu Ile Tyr Thr Leu Pro 1 5 10 15 Ile Leu Gly Leu Leu Gly Leu Leu Thr Ser Pro Ile Leu Thr Lys Phe 20 25 30 Asp Ile Tyr Lys Ile Ser Ile Leu Val Phe Ile Ala Phe Ser Ala Thr 35 40 45 Thr Pro Trp Asp Ser Trp Ile Ile Arg Asn Gly Ala Trp Thr Tyr Pro 50 55 60 Ser Ala Glu Ser Gly Gln Gly Val Phe Gly Thr Phe Leu Asp Val Pro 65 70 75 80 Tyr Glu Glu Tyr Ala Phe Phe Val Ile Gln Thr Val Ile Thr Gly Leu 85 90 95 Val Tyr Val Leu Ala Thr Arg His Leu Leu Pro Ser Leu Ala Leu Pro 100 105 110 Lys Thr Arg Ser Ser Ala Leu Ser Leu Ala Leu Lys Ala Leu Ile Pro 115 120 125 Leu Pro Ile Ile Tyr Leu Phe Thr Ala His Pro Ser Pro Ser Pro Asp 130 135 140 Pro Leu Val Thr Asp His Tyr Phe Tyr Met Arg Ala Leu Ser Leu Leu 145 150 155 160 Ile Thr Pro Pro Thr Met Leu Leu Ala Ala Leu Ser Gly Glu Tyr Ala 165 170 175 Phe Asp Trp Lys Ser Gly Arg Ala Lys Ser Thr Ile Ala Ala Ile Met 180 185 190 Ile Pro Thr Val Tyr Leu Ile Trp Val Asp Tyr Val Ala Val Gly Gln 195 200 205 Asp Ser Trp Ser Ile Asn Asp Glu Lys Ile Val Gly Trp Arg Leu Gly 210 215 220 Gly Val Leu Pro Ile Glu Glu Ala Met Phe Phe Leu Leu Thr Asn Leu 225 230 235 240 Met Ile Val Leu Gly Leu Ser Ala Cys Asp His Thr Gln Ala Leu Tyr 245 250 255 Leu Leu His Gly Arg Thr Ile Tyr Gly Asn Lys Lys Met Pro Ser Ser 260 265 270 Phe Pro Leu Ile Thr Pro Pro Val Leu Ser Leu Phe Phe Ser Ser Arg 275 280 285 Pro Tyr Ser Ser Gln Pro Lys Arg Asp Leu Glu Leu Ala Val Lys Leu 290 295 300 Leu Glu Lys Lys Ser Arg Ser Phe Phe Val Ala Ser Ala Gly Phe Pro 305 310 315 320 Ser Glu Val Arg Glu Arg Leu Val Gly Leu Tyr Ala Phe Cys Arg Val 325 330 335 Thr Asp Asp Leu Ile Asp Ser Pro Glu Val Ser Ser Asn Pro His Ala 340 345 350 Thr Ile Asp Met Val Ser Asp Phe Leu Thr Leu Leu Phe Gly Pro Pro 355 360 365 Leu His Pro Ser Gln Pro Asp Lys Ile Leu Ser Ser Pro Leu Leu Pro 370 375 380 Pro Ser His Pro Ser Arg Pro Thr Gly Met Tyr Pro Leu Pro Pro Pro 385 390 395 400 Pro Ser Leu Ser Pro Ala Glu Leu Val Gln Phe Leu Thr Glu Arg Val 405 410 415 Pro Val Gln Tyr His Phe Ala Phe Arg Leu Leu Ala Lys Leu Gln Gly 420 425 430 Leu Ile Pro Arg Tyr Pro Leu Asp Glu Leu Leu Arg Gly Tyr Thr Thr 435 440 445 Asp Leu Ile Phe Pro Leu Ser Thr Glu Ala Val Gln Ala Arg Lys Thr 450 455 460 Pro Ile Glu Thr Thr Ala Asp Leu Leu Asp Tyr Gly Leu Cys Val Ala 465 470 475 480 Gly Ser Val Ala Glu Leu Leu Val Tyr Val Ser Trp Ala Ser Ala Pro 485 490 495 Ser Gln Val Pro Ala Thr Ile Glu Glu Arg Glu Ala Val Leu Val Ala 500 505 510 Ser Arg Glu Met Gly Thr Ala Leu Gln Leu Val Asn Ile Ala Arg Asp 515 520 525 Ile Lys Gly Asp Ala Thr Glu Gly Arg Phe Tyr Leu Pro Leu Ser Phe 530 535 540 Phe Gly Leu Arg Asp Glu Ser Lys Leu Ala Ile Pro Thr Asp Trp Thr 545 550 555 560 Glu Pro Arg Pro Gln Asp Phe Asp Lys Leu Leu Ser Leu Ser Pro Ser 565 570 575 Ser Thr Leu Pro Ser Ser Asn Ala Ser Glu Ser Phe Arg Phe Glu Trp 580 585 590 Lys Thr Tyr Ser Leu Pro Leu Val Ala Tyr Ala Glu Asp Leu Ala Lys 595 600 605 Ser Tyr Lys Gly Ile Asp Arg Leu Pro Thr Glu Val Gln Ala Gly Met 610 615 620 His Arg Ala Ala Cys Ala Ser Tyr Leu Leu Ile Gly Arg Glu Ile Lys 625 630 635 640 Val Val Trp Lys Gly Asp Val Gly Glu Arg Arg Thr Val Ala Gly Trp 645 650 655 Arg Arg Val Arg Lys Val Leu Ser Val Val Met Ser Gly Trp Glu Gly 660 665 670 Gln 673 570 base pairs nucleic acid double linear cDNA NO NO Phaffia rhodozyma CDS 24..500 /product= “PRcDNA10” 24 AACACTTGGT TAGTTTCGAC GAC ATG CAG ATC TTC GTA AAG ACC CTC ACG 50 Met Gln Ile Phe Val Lys Thr Leu Thr 1 5 GGT AAG ACC ATC ACC CTT GAG GTG GAG TCT TCT GAC ACC ATC GAC AAC 98 Gly Lys Thr Ile Thr Leu Glu Val Glu Ser Ser Asp Thr Ile Asp Asn 10 15 20 25 GTC AAG GCC AAG ATC CAG GAC AAG GAA GGA ATT CCC CCT GAT CAG CAG 146 Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln 30 35 40 CGA CTT ATC TTC GCC GGT AAG CAG CTC GAG GAT GGC CGA ACC CTT TCG 194 Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser 45 50 55 GAT TAC AAC ATC CAG AAA GAG TCC ACC CTC CAC CTC GTC CTT AGG TTG 242 Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu 60 65 70 CGA GGA GGA GCC AAG AAG CGA AAG AAG AAG CAG TAC ACT ACC CCC AAG 290 Arg Gly Gly Ala Lys Lys Arg Lys Lys Lys Gln Tyr Thr Thr Pro Lys 75 80 85 AAG ATC AAG CAC AAG CGA AAG AAG GTC AAG ATG GCT ATT CTT AAG TAC 338 Lys Ile Lys His Lys Arg Lys Lys Val Lys Met Ala Ile Leu Lys Tyr 90 95 100 105 TAC AAG GTC GAC TCT GAT GGA AAG ATC AAG CGA CTT CGT CGA GAG TGC 386 Tyr Lys Val Asp Ser Asp Gly Lys Ile Lys Arg Leu Arg Arg Glu Cys 110 115 120 CCC CAG CCC CAG TGC GGA GCT GGT ATC TTC ATG GCT TTC CAC TCC AAC 434 Pro Gln Pro Gln Cys Gly Ala Gly Ile Phe Met Ala Phe His Ser Asn 125 130 135 CGA CAG ACT TGC GGA AAG TGT GGT CTT ACC TAC ACC TTC GCC GAG GGA 482 Arg Gln Thr Cys Gly Lys Cys Gly Leu Thr Tyr Thr Phe Ala Glu Gly 140 145 150 ACC CAG CCC TCT GCT TAGATCATCA ATCGTTTGTT CCCGAGCGAT CTTTGAGTCT 537 Thr Gln Pro Ser Ala 155 TTGTTACATT CTCAAAAAAA AAAAAAAAAA AAA 570 158 amino acids amino acid linear protein not provided 25 Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Ser Ser Asp Thr Ile Asp Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Ala Lys Lys Arg 65 70 75 80 Lys Lys Lys Gln Tyr Thr Thr Pro Lys Lys Ile Lys His Lys Arg Lys 85 90 95 Lys Val Lys Met Ala Ile Leu Lys Tyr Tyr Lys Val Asp Ser Asp Gly 100 105 110 Lys Ile Lys Arg Leu Arg Arg Glu Cys Pro Gln Pro Gln Cys Gly Ala 115 120 125 Gly Ile Phe Met Ala Phe His Ser Asn Arg Gln Thr Cys Gly Lys Cys 130 135 140 Gly Leu Thr Tyr Thr Phe Ala Glu Gly Thr Gln Pro Ser Ala 145 150 155 303 base pairs nucleic acid double linear cDNA NO NO Phaffia rhodozyma CDS 57..278 /product= “PRcDNA11” 26 TTTACACACA AACCTTACCT ACCTTTTCAA CAACAAATCA CACCTAAGCT TACATC 56 ATG GAG TCC ATC AAG ACC TCG ATT TCC AAC GCC GCC AAC TAC GCT TCT 104 Met Glu Ser Ile Lys Thr Ser Ile Ser Asn Ala Ala Asn Tyr Ala Ser 1 5 10 15 GAG ACT GTC AAC CAG GCC ACT AGC GCC ACC TCC AAG GAG GCC AAC AAG 152 Glu Thr Val Asn Gln Ala Thr Ser Ala Thr Ser Lys Glu Ala Asn Lys 20 25 30 GAG GTT GCC AAG GAC TCC AAT GCC GGA GTT GGA ACC CGA ATC AAC GCC 200 Glu Val Ala Lys Asp Ser Asn Ala Gly Val Gly Thr Arg Ile Asn Ala 35 40 45 GGA ATT GAT GCT CTT GGA GAC AAG GCC GAC GAG ACT TCG TCT GAT GCC 248 Gly Ile Asp Ala Leu Gly Asp Lys Ala Asp Glu Thr Ser Ser Asp Ala 50 55 60 AAG TCC AAG GCC TAC AAG CAG AAC ATC TAAGTTATTT AGATAGTCGT 295 Lys Ser Lys Ala Tyr Lys Gln Asn Ile 65 70 CCATATTT 303 73 amino acids amino acid linear protein not provided 27 Met Glu Ser Ile Lys Thr Ser Ile Ser Asn Ala Ala Asn Tyr Ala Ser 1 5 10 15 Glu Thr Val Asn Gln Ala Thr Ser Ala Thr Ser Lys Glu Ala Asn Lys 20 25 30 Glu Val Ala Lys Asp Ser Asn Ala Gly Val Gly Thr Arg Ile Asn Ala 35 40 45 Gly Ile Asp Ala Leu Gly Asp Lys Ala Asp Glu Thr Ser Ser Asp Ala 50 55 60 Lys Ser Lys Ala Tyr Lys Gln Asn Ile 65 70 307 base pairs nucleic acid double linear cDNA NO NO Phaffia rhodozyma CDS 3..227 /product= “PRcDNA18” 28 AC CCT TCC ATC GAG TCT GAG GCC CGA CAA CAC AAG CTC AAG AGG CTT 47 Pro Ser Ile Glu Ser Glu Ala Arg Gln His Lys Leu Lys Arg Leu 1 5 10 15 GTG CAG AGC CCC AAC TCT TTC TTC ATG GAC GTC AAG TGC CCT GGT TGC 95 Val Gln Ser Pro Asn Ser Phe Phe Met Asp Val Lys Cys Pro Gly Cys 20 25 30 TTC CAG ATC ACC ACC GTG TTC TCG CAC GCT TCC ACT GCC GTT CAG TGT 143 Phe Gln Ile Thr Thr Val Phe Ser His Ala Ser Thr Ala Val Gln Cys 35 40 45 GGA TCG TGC CAG ACC ATC CTC TGC CAG CCC CGG GGA GGA AAG GCT CGA 191 Gly Ser Cys Gln Thr Ile Leu Cys Gln Pro Arg Gly Gly Lys Ala Arg 50 55 60 CTT ACC GAG GGA TGC TCT TTC CGA CGA AAG AAC TAAGTTTCTG TTATCGGATG 244 Leu Thr Glu Gly Cys Ser Phe Arg Arg Lys Asn 65 70 75 ATGCATTCAA ATAAAAGTCA AAAAAAAAAA AAAAAAAAAC TCGAGGGGGG GCCCGGTACC 304 CAA 307 74 amino acids amino acid linear protein not provided 29 Pro Ser Ile Glu Ser Glu Ala Arg Gln His Lys Leu Lys Arg Leu Val 1 5 10 15 Gln Ser Pro Asn Ser Phe Phe Met Asp Val Lys Cys Pro Gly Cys Phe 20 25 30 Gln Ile Thr Thr Val Phe Ser His Ala Ser Thr Ala Val Gln Cys Gly 35 40 45 Ser Cys Gln Thr Ile Leu Cys Gln Pro Arg Gly Gly Lys Ala Arg Leu 50 55 60 Thr Glu Gly Cys Ser Phe Arg Arg Lys Asn 65 70 502 base pairs nucleic acid double linear cDNA NO NO Phaffia rhodozyma CDS 30..359 /product= “PRcDNA35” 30 GTCAGCTCCG GCTTAAATCG ATTCGTACA ATG TCT GAA CTC GCC GCC TCC TAC 53 Met Ser Glu Leu Ala Ala Ser Tyr 1 5 GCC GCT CTT ATC CTC GCC GAC GAG GGT ATT GAG ATC ACC TCT GAG AAG 101 Ala Ala Leu Ile Leu Ala Asp Glu Gly Ile Glu Ile Thr Ser Glu Lys 10 15 20 CTC GTC ACT CTC ACC ACC GCC GCC AAG GTT GAG CTT GAG CCC ATC TGG 149 Leu Val Thr Leu Thr Thr Ala Ala Lys Val Glu Leu Glu Pro Ile Trp 25 30 35 40 GCC ACT CTC CTT GCC AAG GCC CTC GAG GGA AAG AAC GTC AAG GAG TTG 197 Ala Thr Leu Leu Ala Lys Ala Leu Glu Gly Lys Asn Val Lys Glu Leu 45 50 55 CTT TCC AAC GTC GGA TCC GGA GCC GGA GGA GCT GCC CCC GCC GCC GCC 245 Leu Ser Asn Val Gly Ser Gly Ala Gly Gly Ala Ala Pro Ala Ala Ala 60 65 70 GTC GCC GGT GGA GCT TCC GCT GAC GCC TCT GCC CCC GCT GAG GAG AAG 293 Val Ala Gly Gly Ala Ser Ala Asp Ala Ser Ala Pro Ala Glu Glu Lys 75 80 85 AAG GAG GAG AAG GCT GAG GAC AAG GAG GAG TCT GAC GAC GAC ATG GGT 341 Lys Glu Glu Lys Ala Glu Asp Lys Glu Glu Ser Asp Asp Asp Met Gly 90 95 100 TTC GGA CTT TTC GAT TAAACTCCCT CGCCTAAAAA CCCTTTTCTT CAACCCCCTC 396 Phe Gly Leu Phe Asp 105 TCGTGGCATC GTTCACTCGA CCGCTGCGTT TGTTGTCCTT TCCTCACGAA TTTTGTCCTT 456 GTCTGGTTTC CCAATNGGAT NTCCTTGAAA TGANGTTTCC CAATTG 502 109 amino acids amino acid linear protein not provided 31 Met Ser Glu Leu Ala Ala Ser Tyr Ala Ala Leu Ile Leu Ala Asp Glu 1 5 10 15 Gly Ile Glu Ile Thr Ser Glu Lys Leu Val Thr Leu Thr Thr Ala Ala 20 25 30 Lys Val Glu Leu Glu Pro Ile Trp Ala Thr Leu Leu Ala Lys Ala Leu 35 40 45 Glu Gly Lys Asn Val Lys Glu Leu Leu Ser Asn Val Gly Ser Gly Ala 50 55 60 Gly Gly Ala Ala Pro Ala Ala Ala Val Ala Gly Gly Ala Ser Ala Asp 65 70 75 80 Ala Ser Ala Pro Ala Glu Glu Lys Lys Glu Glu Lys Ala Glu Asp Lys 85 90 95 Glu Glu Ser Asp Asp Asp Met Gly Phe Gly Leu Phe Asp 100 105 381 base pairs nucleic acid double linear cDNA NO NO Phaffia rhodozyma CDS 7..282 /product= “PRcDNA38” 32 CTCAAG ATG ACC AAA GGT ACC TCC TCT TTC GGT AAG CGA CAC ACC AAG 48 Met Thr Lys Gly Thr Ser Ser Phe Gly Lys Arg His Thr Lys 1 5 10 ACC CAC ACC ATC TGC CGA CGA TGT GGT AAC AGG GCT TTC CAC AGG CAG 96 Thr His Thr Ile Cys Arg Arg Cys Gly Asn Arg Ala Phe His Arg Gln 15 20 25 30 AAG AAG ACC TGT GCC CAG TGT GGA TAC CCT GCC GCC AAG ATG CGA AGC 144 Lys Lys Thr Cys Ala Gln Cys Gly Tyr Pro Ala Ala Lys Met Arg Ser 35 40 45 TTC AAC TGG GGA GAG AAG GCC AAG AGG AGA AAG ACC ACC GGT ACC GGT 192 Phe Asn Trp Gly Glu Lys Ala Lys Arg Arg Lys Thr Thr Gly Thr Gly 50 55 60 CGA ATG CAG CAC CTC AAG GAC GTC TCT CGA CGA TTC AAG AAC GGC TTC 240 Arg Met Gln His Leu Lys Asp Val Ser Arg Arg Phe Lys Asn Gly Phe 65 70 75 CGA GAG GGA ACT TCC GCC ACC AAG AAG GTC AAG GCC GAG TAATCGGTTT 289 Arg Glu Gly Thr Ser Ala Thr Lys Lys Val Lys Ala Glu 80 85 90 ATCCATCACC TGGTGATCAG GGCGGGTAAT AATCTTTTGT TAGAGACTAT CCATGTTCTG 349 CTGCCGCATC AAACAAAAAA AAAAAAAAAA AA 381 91 amino acids amino acid linear protein not provided 33 Met Thr Lys Gly Thr Ser Ser Phe Gly Lys Arg His Thr Lys Thr His 1 5 10 15 Thr Ile Cys Arg Arg Cys Gly Asn Arg Ala Phe His Arg Gln Lys Lys 20 25 30 Thr Cys Ala Gln Cys Gly Tyr Pro Ala Ala Lys Met Arg Ser Phe Asn 35 40 45 Trp Gly Glu Lys Ala Lys Arg Arg Lys Thr Thr Gly Thr Gly Arg Met 50 55 60 Gln His Leu Lys Asp Val Ser Arg Arg Phe Lys Asn Gly Phe Arg Glu 65 70 75 80 Gly Thr Ser Ala Thr Lys Lys Val Lys Ala Glu 85 90 473 base pairs nucleic acid double linear cDNA NO NO Phaffia rhodozyma CDS 19..321 /product= “PRcDNA46” 34 CTCAAGAAGA AACTCGCC ATG CCT ACC CGA TTC TCC AAC ACC CGA AAG CAC 51 Met Pro Thr Arg Phe Ser Asn Thr Arg Lys His 1 5 10 AGA GGA CAC GTC TCT GCC GGT CAC GGT CGT GTG GGA AAG CAC AGA AAG 99 Arg Gly His Val Ser Ala Gly His Gly Arg Val Gly Lys His Arg Lys 15 20 25 CAC CCA GGA GGA CGA GGT CTT GCT GGA GGA CAG CAC CAC CAC CGA ACC 147 His Pro Gly Gly Arg Gly Leu Ala Gly Gly Gln His His His Arg Thr 30 35 40 AAC TTC GAT AAG TAC CAC CCT GGA TAC TTC GGA AAG GTC GGA ATG AGG 195 Asn Phe Asp Lys Tyr His Pro Gly Tyr Phe Gly Lys Val Gly Met Arg 45 50 55 CAC TTC CAC CTT ACC CGA NAC TCT TCC TGG TGC CCT ACC GTC AAC ATT 243 His Phe His Leu Thr Arg Xaa Ser Ser Trp Cys Pro Thr Val Asn Ile 60 65 70 75 GAC NAG CTC TGG ACT CTC GTC CCC GCT GAG GAG AAG AAG GAC TTC CCC 291 Asp Xaa Leu Trp Thr Leu Val Pro Ala Glu Glu Lys Lys Asp Phe Pro 80 85 90 AAC CAG GCT CGA CCT CGT CCC CGT TGT TGACACTTTG GCTCTCGGTT 338 Asn Gln Ala Arg Pro Arg Pro Arg Cys 95 100 ACGGCAATGT TCTTGGCAAG GGTCTACTTC CCCAGATCCC TTTAATCGTC AAGGCCCGAT 398 TCNTTTCCGC TCTTGCCGAG AANAANATCN ANGANGCTGG TTGGAATTCC TCTCCCCTTT 458 GTTCCCCCCN TAANG 473 100 amino acids amino acid linear protein not provided 35 Met Pro Thr Arg Phe Ser Asn Thr Arg Lys His Arg Gly His Val Ser 1 5 10 15 Ala Gly His Gly Arg Val Gly Lys His Arg Lys His Pro Gly Gly Arg 20 25 30 Gly Leu Ala Gly Gly Gln His His His Arg Thr Asn Phe Asp Lys Tyr 35 40 45 His Pro Gly Tyr Phe Gly Lys Val Gly Met Arg His Phe His Leu Thr 50 55 60 Arg Xaa Ser Ser Trp Cys Pro Thr Val Asn Ile Asp Xaa Leu Trp Thr 65 70 75 80 Leu Val Pro Ala Glu Glu Lys Lys Asp Phe Pro Asn Gln Ala Arg Pro 85 90 95 Arg Pro Arg Cys 100 608 base pairs nucleic acid double linear cDNA NO NO Phaffia rhodozyma CDS 18..453 /product= “PRcDNA64” 36 AAGACTCGTC GTTCAGC ATG TCC TCC GTC AAA GCC ACC AAA GGA AAG GGT 50 Met Ser Ser Val Lys Ala Thr Lys Gly Lys Gly 1 5 10 CCC GCC GCC TCG GCT GAT GTT AAG GCC AAG GCC GCC AAG AAG GCT GCC 98 Pro Ala Ala Ser Ala Asp Val Lys Ala Lys Ala Ala Lys Lys Ala Ala 15 20 25 CTC AAG GGT ACT CAG TCT ACT TCC ACC AGG AAG GTC CGA ACT TCG GTC 146 Leu Lys Gly Thr Gln Ser Thr Ser Thr Arg Lys Val Arg Thr Ser Val 30 35 40 TCT TTC CAC CGA CCC AAG ACT CTC CGA CTT CCC CGA GCT CCC AAG TAC 194 Ser Phe His Arg Pro Lys Thr Leu Arg Leu Pro Arg Ala Pro Lys Tyr 45 50 55 CCC CGA AAG TCG GTC CCT CAC GCC CCT CGA ATG GAT GAG TTC CGA ACT 242 Pro Arg Lys Ser Val Pro His Ala Pro Arg Met Asp Glu Phe Arg Thr 60 65 70 75 ATC ATC CAC CCC TTG GCT ACC GAG TCC GCC ATG AAG AAG ATT GAG GAG 290 Ile Ile His Pro Leu Ala Thr Glu Ser Ala Met Lys Lys Ile Glu Glu 80 85 90 CAC AAC ACC CTT GTG TTC ATC GTC GAT GTC AAG TCC AAC AAG CGA CAG 338 His Asn Thr Leu Val Phe Ile Val Asp Val Lys Ser Asn Lys Arg Gln 95 100 105 ATC AAG GAC GCC GTC AAG AAG CTC TAC GAG GTC GAT ACC GTC CAC NTC 386 Ile Lys Asp Ala Val Lys Lys Leu Tyr Glu Val Asp Thr Val His Xaa 110 115 120 AAC NCC TTG ATC ACC CCC GCC GGA AGG AAG AAG CTT ACG TCC GAC TTA 434 Asn Xaa Leu Ile Thr Pro Ala Gly Arg Lys Lys Leu Thr Ser Asp Leu 125 130 135 CCC CCG ACC ACG ACG CTC T TAACGTTGCC AACAAGGCCG GCTACATCTA 483 Pro Pro Thr Thr Thr Leu 140 145 ATCGACTCCA TCCCTTGGAT CGGTTCAGTT GTTTGGTTTG CATCCGGTTT CAGAGTTTGA 543 CGACCTTGAA ACTCNAANAC TTTGGATGCA TGTTTGAAAT TCTCNAAATA AAAAAAAAAA 603 AAAAA 608 145 amino acids amino acid linear protein not provided 37 Met Ser Ser Val Lys Ala Thr Lys Gly Lys Gly Pro Ala Ala Ser Ala 1 5 10 15 Asp Val Lys Ala Lys Ala Ala Lys Lys Ala Ala Leu Lys Gly Thr Gln 20 25 30 Ser Thr Ser Thr Arg Lys Val Arg Thr Ser Val Ser Phe His Arg Pro 35 40 45 Lys Thr Leu Arg Leu Pro Arg Ala Pro Lys Tyr Pro Arg Lys Ser Val 50 55 60 Pro His Ala Pro Arg Met Asp Glu Phe Arg Thr Ile Ile His Pro Leu 65 70 75 80 Ala Thr Glu Ser Ala Met Lys Lys Ile Glu Glu His Asn Thr Leu Val 85 90 95 Phe Ile Val Asp Val Lys Ser Asn Lys Arg Gln Ile Lys Asp Ala Val 100 105 110 Lys Lys Leu Tyr Glu Val Asp Thr Val His Xaa Asn Xaa Leu Ile Thr 115 120 125 Pro Ala Gly Arg Lys Lys Leu Thr Ser Asp Leu Pro Pro Thr Thr Thr 130 135 140 Leu 145 466 base pairs nucleic acid double linear cDNA NO NO Phaffia rhodozyma CDS 81..416 /product= “PRcDNA68” 38 CTTTGAACCT CCAACCTCGG CATCAAGCAC TAGTCAGCCT CGGCTTAAAT CGATTCGTGT 60 AGCCTTTCAA ACTCGTAAAA ATG AAG CAC ATC GCC GCT TAC TTG CTC CTC 110 Met Lys His Ile Ala Ala Tyr Leu Leu Leu 1 5 10 GCC ACC GGT GGA AAC NCC TCC CCC TCT GCC GCC GAT GTC AAG GCC CTC 158 Ala Thr Gly Gly Asn Xaa Ser Pro Ser Ala Ala Asp Val Lys Ala Leu 15 20 25 CTT GCC ACC GTC GAC ATC GAG GCT GAT GAC GCC CGA CTT GAG ACC CTC 206 Leu Ala Thr Val Asp Ile Glu Ala Asp Asp Ala Arg Leu Glu Thr Leu 30 35 40 ATC TCC GAG CTT AAC GGC AAG GAC TTG AAC ACC CTC ATC GCT GAG GGA 254 Ile Ser Glu Leu Asn Gly Lys Asp Leu Asn Thr Leu Ile Ala Glu Gly 45 50 55 TCC GCC AAG CTC GCT TCC GTC CCC TCC GGA GGA GCC GCC TCT TCC GCT 302 Ser Ala Lys Leu Ala Ser Val Pro Ser Gly Gly Ala Ala Ser Ser Ala 60 65 70 GCC CCC GCC GCC GCT GGA GGA GCC GCC GCC CCT GCC GCT GAG GAT AAG 350 Ala Pro Ala Ala Ala Gly Gly Ala Ala Ala Pro Ala Ala Glu Asp Lys 75 80 85 90 AAG GAG GAG AAG GTC GAG GAC AAG GAG GAG TCT GAC GAC GAC ATG GGT 398 Lys Glu Glu Lys Val Glu Asp Lys Glu Glu Ser Asp Asp Asp Met Gly 95 100 105 TTC GGA CTT TTC GAT TAAACTCCTT ACACCTTTTT CAAACTCTTC GTTGGCTCGA 453 Phe Gly Leu Phe Asp 110 GGGGGGGCCC GGT 466 111 amino acids amino acid linear protein not provided 39 Met Lys His Ile Ala Ala Tyr Leu Leu Leu Ala Thr Gly Gly Asn Xaa 1 5 10 15 Ser Pro Ser Ala Ala Asp Val Lys Ala Leu Leu Ala Thr Val Asp Ile 20 25 30 Glu Ala Asp Asp Ala Arg Leu Glu Thr Leu Ile Ser Glu Leu Asn Gly 35 40 45 Lys Asp Leu Asn Thr Leu Ile Ala Glu Gly Ser Ala Lys Leu Ala Ser 50 55 60 Val Pro Ser Gly Gly Ala Ala Ser Ser Ala Ala Pro Ala Ala Ala Gly 65 70 75 80 Gly Ala Ala Ala Pro Ala Ala Glu Asp Lys Lys Glu Glu Lys Val Glu 85 90 95 Asp Lys Glu Glu Ser Asp Asp Asp Met Gly Phe Gly Leu Phe Asp 100 105 110 570 base pairs nucleic acid double linear cDNA NO NO Phaffia rhodozyma CDS 49..501 /product= “PRcDNA73” 40 CTTCCTCCCG TCAAGGCAAA CCTTCAGAAT CCTCTCAAGT CATTCAAC ATG GGA CGA 57 Met Gly Arg 1 GTC CGC ACC AAA ACC GTC AAG CGA GCT TCG CGA GTG ATG ATC GAG AAG 105 Val Arg Thr Lys Thr Val Lys Arg Ala Ser Arg Val Met Ile Glu Lys 5 10 15 TTC TAC CCT CGA CTC ACT CTT GAT TTC CAC ACC AAC AAG CGA ATC GCC 153 Phe Tyr Pro Arg Leu Thr Leu Asp Phe His Thr Asn Lys Arg Ile Ala 20 25 30 35 GAC GAG GTT GCC ATC ATC CCC TCC AAG CGA CTT CGA AAC AAG ATC GCT 201 Asp Glu Val Ala Ile Ile Pro Ser Lys Arg Leu Arg Asn Lys Ile Ala 40 45 50 GGG TTC ACT ACC CAC TTG ATG AAG CGA ATC CAG AAG GGA CCC GTT CGA 249 Gly Phe Thr Thr His Leu Met Lys Arg Ile Gln Lys Gly Pro Val Arg 55 60 65 GGT ATC TCC TTC AAG CTT CAG GAG GAG GAG CGA GAG AGG AAG GAT CAG 297 Gly Ile Ser Phe Lys Leu Gln Glu Glu Glu Arg Glu Arg Lys Asp Gln 70 75 80 TAC GTT CCT GAG GTC TCC GCC CTT GCC GCC CCT GAG CTG GGT TTG GAG 345 Tyr Val Pro Glu Val Ser Ala Leu Ala Ala Pro Glu Leu Gly Leu Glu 85 90 95 GTT GAC CCC GAC ACC AAG GAT CTT CTC CGA TCC CTT GGC ATG GAC TCC 393 Val Asp Pro Asp Thr Lys Asp Leu Leu Arg Ser Leu Gly Met Asp Ser 100 105 110 115 ATC AAC GTC CAG GTC TCC GCT CCT ATC TCT TCC TAC GCT GCC CCC GAG 441 Ile Asn Val Gln Val Ser Ala Pro Ile Ser Ser Tyr Ala Ala Pro Glu 120 125 130 CGA GGT CCC CGA GGT GCC GGA CGA NGT GGA CGA ATC GTC CCC GGA GCT 489 Arg Gly Pro Arg Gly Ala Gly Arg Xaa Gly Arg Ile Val Pro Gly Ala 135 140 145 GGC CGA TAC TAAGTGTTTT CTTCAACCAN GGGATATTTG ATNATTCGCT 538 Gly Arg Tyr 150 AGGCTTGAAA TTTTTTTATC ATTCTTCCTA TA 570 150 amino acids amino acid linear protein not provided 41 Met Gly Arg Val Arg Thr Lys Thr Val Lys Arg Ala Ser Arg Val Met 1 5 10 15 Ile Glu Lys Phe Tyr Pro Arg Leu Thr Leu Asp Phe His Thr Asn Lys 20 25 30 Arg Ile Ala Asp Glu Val Ala Ile Ile Pro Ser Lys Arg Leu Arg Asn 35 40 45 Lys Ile Ala Gly Phe Thr Thr His Leu Met Lys Arg Ile Gln Lys Gly 50 55 60 Pro Val Arg Gly Ile Ser Phe Lys Leu Gln Glu Glu Glu Arg Glu Arg 65 70 75 80 Lys Asp Gln Tyr Val Pro Glu Val Ser Ala Leu Ala Ala Pro Glu Leu 85 90 95 Gly Leu Glu Val Asp Pro Asp Thr Lys Asp Leu Leu Arg Ser Leu Gly 100 105 110 Met Asp Ser Ile Asn Val Gln Val Ser Ala Pro Ile Ser Ser Tyr Ala 115 120 125 Ala Pro Glu Arg Gly Pro Arg Gly Ala Gly Arg Xaa Gly Arg Ile Val 130 135 140 Pro Gly Ala Gly Arg Tyr 145 150 373 base pairs nucleic acid double linear cDNA NO NO Phaffia rhodozyma CDS 13..324 /product= “PRcDNA76” 42 CCATCATCCA AC ATG CCT CCC AAA GTC AAG GCC AAG ACC GGT GTC GGT 48 Met Pro Pro Lys Val Lys Ala Lys Thr Gly Val Gly 1 5 10 AAG ACC CAG AAG AAG AAG AAG TGG TCC AAG GGA AAG GTG AAG GAC AAG 96 Lys Thr Gln Lys Lys Lys Lys Trp Ser Lys Gly Lys Val Lys Asp Lys 15 20 25 GCC GCC CAC CAC GTC GTT GTT GAT CAG GCC ACT TAC GAC AAG ATC GTT 144 Ala Ala His His Val Val Val Asp Gln Ala Thr Tyr Asp Lys Ile Val 30 35 40 AAG GAG GTC CCC ACC TAC AAG TTG ATC TCC CAG TCT ATC TTG ATT GAC 192 Lys Glu Val Pro Thr Tyr Lys Leu Ile Ser Gln Ser Ile Leu Ile Asp 45 50 55 60 CGA CAC AAG GTT AAC GGT TCC GTC GCC CGA GCC GCT ATC CGA CAC CTT 240 Arg His Lys Val Asn Gly Ser Val Ala Arg Ala Ala Ile Arg His Leu 65 70 75 GCC AAG GAG GGA TCC ATC AAG AAG ATT GTC CAC CAC AAC GGA CAG TGG 288 Ala Lys Glu Gly Ser Ile Lys Lys Ile Val His His Asn Gly Gln Trp 80 85 90 ATC TAC ACC CGA GCC ACT GCC GCT CCT GAC GCA TAAATCTGAT GGATTTCATG 341 Ile Tyr Thr Arg Ala Thr Ala Ala Pro Asp Ala 95 100 GATCTTGAAA AATAAAAAAA AAAAAAAAAA AA 373 103 amino acids amino acid linear protein not provided 43 Met Pro Pro Lys Val Lys Ala Lys Thr Gly Val Gly Lys Thr Gln Lys 1 5 10 15 Lys Lys Lys Trp Ser Lys Gly Lys Val Lys Asp Lys Ala Ala His His 20 25 30 Val Val Val Asp Gln Ala Thr Tyr Asp Lys Ile Val Lys Glu Val Pro 35 40 45 Thr Tyr Lys Leu Ile Ser Gln Ser Ile Leu Ile Asp Arg His Lys Val 50 55 60 Asn Gly Ser Val Ala Arg Ala Ala Ile Arg His Leu Ala Lys Glu Gly 65 70 75 80 Ser Ile Lys Lys Ile Val His His Asn Gly Gln Trp Ile Tyr Thr Arg 85 90 95 Ala Thr Ala Ala Pro Asp Ala 100 514 base pairs nucleic acid double linear cDNA NO NO Phaffia rhodozyma CDS 13..435 /product= “PRcDNA78” 44 AAAAAAGCCA AT ATG CTT ATC TCT AAA CAG AAC AGG AGG GCC ATC TTC 48 Met Leu Ile Ser Lys Gln Asn Arg Arg Ala Ile Phe 1 5 10 GAG AAC CTC TTC AAG GAG GGA GTT GCC GTC GCC GCC AAG GAC TTC AAC 96 Glu Asn Leu Phe Lys Glu Gly Val Ala Val Ala Ala Lys Asp Phe Asn 15 20 25 GCT GCC ACC CAC CCC GAG ATT GAG GGT GTC TCC AAC CTT GAG GTC ATC 144 Ala Ala Thr His Pro Glu Ile Glu Gly Val Ser Asn Leu Glu Val Ile 30 35 40 AAG GCC ATG CAG TCT TTG ACC TCC AAG GGA TAC GTG AAG ACC CAG TTC 192 Lys Ala Met Gln Ser Leu Thr Ser Lys Gly Tyr Val Lys Thr Gln Phe 45 50 55 60 TCG TGG CAG TAC TAC TAC TAC ACC CTC ACC CCT GAG GGT CTT GAC TAC 240 Ser Trp Gln Tyr Tyr Tyr Tyr Thr Leu Thr Pro Glu Gly Leu Asp Tyr 65 70 75 CTC CGA GAG TTC CTC CAC CTT CCC TCC GAG ATT GTC CCC AAC ACT CTC 288 Leu Arg Glu Phe Leu His Leu Pro Ser Glu Ile Val Pro Asn Thr Leu 80 85 90 AAG CGA CCC ACC CGA CCT GCC AAG GCC CAG GGT CCC GGA GGT GCC TAC 336 Lys Arg Pro Thr Arg Pro Ala Lys Ala Gln Gly Pro Gly Gly Ala Tyr 95 100 105 CGA GCT CCC CGA GCC GAG GGT GCC GGT CGA GGA GAG TAC CGA CGA CGA 384 Arg Ala Pro Arg Ala Glu Gly Ala Gly Arg Gly Glu Tyr Arg Arg Arg 110 115 120 GAG GAC GGT GCC GGT GCC TTC GGT GCC GGT CGA GGT GGA CCC CGA GCT 432 Glu Asp Gly Ala Gly Ala Phe Gly Ala Gly Arg Gly Gly Pro Arg Ala 125 130 135 140 TAAATCCCAG AGCTTTTCTT TTTGTCGTTG CTGGGACTAT GGCATGATGA GCTGGCTTGC 492 AGAAAAAAAA AAAAAAAAAA AA 514 140 amino acids amino acid linear protein not provided 45 Met Leu Ile Ser Lys Gln Asn Arg Arg Ala Ile Phe Glu Asn Leu Phe 1 5 10 15 Lys Glu Gly Val Ala Val Ala Ala Lys Asp Phe Asn Ala Ala Thr His 20 25 30 Pro Glu Ile Glu Gly Val Ser Asn Leu Glu Val Ile Lys Ala Met Gln 35 40 45 Ser Leu Thr Ser Lys Gly Tyr Val Lys Thr Gln Phe Ser Trp Gln Tyr 50 55 60 Tyr Tyr Tyr Thr Leu Thr Pro Glu Gly Leu Asp Tyr Leu Arg Glu Phe 65 70 75 80 Leu His Leu Pro Ser Glu Ile Val Pro Asn Thr Leu Lys Arg Pro Thr 85 90 95 Arg Pro Ala Lys Ala Gln Gly Pro Gly Gly Ala Tyr Arg Ala Pro Arg 100 105 110 Ala Glu Gly Ala Gly Arg Gly Glu Tyr Arg Arg Arg Glu Asp Gly Ala 115 120 125 Gly Ala Phe Gly Ala Gly Arg Gly Gly Pro Arg Ala 130 135 140 437 base pairs nucleic acid double linear cDNA NO NO Phaffia rhodozyma CDS 30..308 /product= “PRcDNA85” 46 CTCCCTCAAG AAATCAACCA CCGCACATC ATG TCC AAG CGA ACC AAG AAA GTT 53 Met Ser Lys Arg Thr Lys Lys Val 1 5 GGA ATC ACC GGA AAG TAC GGA GTC CGA TAC GGA GCT TCC CTC CGA AAG 101 Gly Ile Thr Gly Lys Tyr Gly Val Arg Tyr Gly Ala Ser Leu Arg Lys 10 15 20 ACC GTC AAG AAG NTG GAG GTC TGG CAG CAC GGT ACC TAC ACC TGT GAC 149 Thr Val Lys Lys Xaa Glu Val Trp Gln His Gly Thr Tyr Thr Cys Asp 25 30 35 40 TTC TGC GGA AAG GAC GCC GTC AAG CGA ACC GCT GTT GGT ATC TGG AAG 197 Phe Cys Gly Lys Asp Ala Val Lys Arg Thr Ala Val Gly Ile Trp Lys 45 50 55 TGC CGA GGA TGC CGA AAG ACC ACC GCC GGT GGT GCT TGG CAG CTT CAG 245 Cys Arg Gly Cys Arg Lys Thr Thr Ala Gly Gly Ala Trp Gln Leu Gln 60 65 70 ACC ACC GCC GCT CTC ACC GTC AAG TCC ACC ACT CGA CGA CTC CGA GAG 293 Thr Thr Ala Ala Leu Thr Val Lys Ser Thr Thr Arg Arg Leu Arg Glu 75 80 85 CTC AAG GAG GTT TAAATTGAAT TCTGCACAAA GACAAAACTG TTGCGGGCGG 345 Leu Lys Glu Val 90 GAGAGAGTGG ATTCATTCTT TTTTTTTGTA GATCTGAAGG GATGCCATGT CAACCCTTTC 405 GTTCCCCAAA AAAAAAAAAA AAAAAAAAAA AA 437 92 amino acids amino acid linear protein not provided 47 Met Ser Lys Arg Thr Lys Lys Val Gly Ile Thr Gly Lys Tyr Gly Val 1 5 10 15 Arg Tyr Gly Ala Ser Leu Arg Lys Thr Val Lys Lys Xaa Glu Val Trp 20 25 30 Gln His Gly Thr Tyr Thr Cys Asp Phe Cys Gly Lys Asp Ala Val Lys 35 40 45 Arg Thr Ala Val Gly Ile Trp Lys Cys Arg Gly Cys Arg Lys Thr Thr 50 55 60 Ala Gly Gly Ala Trp Gln Leu Gln Thr Thr Ala Ala Leu Thr Val Lys 65 70 75 80 Ser Thr Thr Arg Arg Leu Arg Glu Leu Lys Glu Val 85 90 509 base pairs nucleic acid double linear cDNA NO NO Phaffia rhodozyma CDS 35..400 /product= “PRcDNA87” 48 GGAAGACCTC ACAGCAAGAC TAAGACTCTC AAAC ATG GCT ACC AAG ACC GGC 52 Met Ala Thr Lys Thr Gly 1 5 AAG ACT CGA TCC GCT CTC CAG GAC GTC GTT ACT CGG GAG TAC ACC ATC 100 Lys Thr Arg Ser Ala Leu Gln Asp Val Val Thr Arg Glu Tyr Thr Ile 10 15 20 CAC CTC CAC AAG TAC GTT CAC GGA AGG TCT TTC AAG AAG CGA GCT CCT 148 His Leu His Lys Tyr Val His Gly Arg Ser Phe Lys Lys Arg Ala Pro 25 30 35 TGG GCT GTC AAG TCC ATC CAG GAG TTT GCT CTC AAG TCG ATG GGA ACC 196 Trp Ala Val Lys Ser Ile Gln Glu Phe Ala Leu Lys Ser Met Gly Thr 40 45 50 CGA GAT GTC CGA ATT GAC CCC AAG TTG AAC CAG GCC GTC TGG GGA CAG 244 Arg Asp Val Arg Ile Asp Pro Lys Leu Asn Gln Ala Val Trp Gly Gln 55 60 65 70 GGT GTC AAG AAC CCC CCC AAG CGA CTC CGA ATC CGA CTT GAG CGA AAG 292 Gly Val Lys Asn Pro Pro Lys Arg Leu Arg Ile Arg Leu Glu Arg Lys 75 80 85 CGA AAC GAC GAG GAG GAT GCT AAG GAC AAG CTC TAC ACT CTT GCT ACC 340 Arg Asn Asp Glu Glu Asp Ala Lys Asp Lys Leu Tyr Thr Leu Ala Thr 90 95 100 GTC GTC CCC GGA GTC ACC AAC TTC AAG GGT CTC CAA ACC GTT GTC GTT 388 Val Val Pro Gly Val Thr Asn Phe Lys Gly Leu Gln Thr Val Val Val 105 110 115 GAC ACC GAG TAATTTTGTC TTGGATTTTC ATGACGGTCG ATTCAGCTGT 437 Asp Thr Glu 120 TTCTTGGCGC CATTCTTCTT ATGCACTCTG ATGCCTTTCA CGACCCNTTT TTNTTTCTNA 497 TAAATAAAAA AA 509 121 amino acids amino acid linear protein not provided 49 Met Ala Thr Lys Thr Gly Lys Thr Arg Ser Ala Leu Gln Asp Val Val 1 5 10 15 Thr Arg Glu Tyr Thr Ile His Leu His Lys Tyr Val His Gly Arg Ser 20 25 30 Phe Lys Lys Arg Ala Pro Trp Ala Val Lys Ser Ile Gln Glu Phe Ala 35 40 45 Leu Lys Ser Met Gly Thr Arg Asp Val Arg Ile Asp Pro Lys Leu Asn 50 55 60 Gln Ala Val Trp Gly Gln Gly Val Lys Asn Pro Pro Lys Arg Leu Arg 65 70 75 80 Ile Arg Leu Glu Arg Lys Arg Asn Asp Glu Glu Asp Ala Lys Asp Lys 85 90 95 Leu Tyr Thr Leu Ala Thr Val Val Pro Gly Val Thr Asn Phe Lys Gly 100 105 110 Leu Gln Thr Val Val Val Asp Thr Glu 115 120 542 base pairs nucleic acid double linear cDNA NO NO Phaffia rhodozyma CDS 18..443 /product= “PRcDNA95” 50 AGTCGCTATA CATCAAG ATG TCC GTC GCT GTC CAG ACT TTC GGT AAG AAG 50 Met Ser Val Ala Val Gln Thr Phe Gly Lys Lys 1 5 10 AAG ACT GCC ACC GCT GTG GCC CAC GCC ACC CCT GGC CGA GGT CTC ATC 98 Lys Thr Ala Thr Ala Val Ala His Ala Thr Pro Gly Arg Gly Leu Ile 15 20 25 CGA CTT AAC GGA CAG CCT ATC TCA CTT GCC GAG CCT GCT CTC CTC CGA 146 Arg Leu Asn Gly Gln Pro Ile Ser Leu Ala Glu Pro Ala Leu Leu Arg 30 35 40 TAC AAG TAC TAC GAG CCT ATC CTC GTC ATC GGA GCT GAG AAG ATC AAC 194 Tyr Lys Tyr Tyr Glu Pro Ile Leu Val Ile Gly Ala Glu Lys Ile Asn 45 50 55 CAG ATC GAC ATC CGA CTC AAG GTC AAG GGT GGA GGA CAC GTC TCC CAG 242 Gln Ile Asp Ile Arg Leu Lys Val Lys Gly Gly Gly His Val Ser Gln 60 65 70 75 GTG TAC GCC GTC CGA CAG GCC ATC GGT AAG GCC ATC GTC GCT TAC TAC 290 Val Tyr Ala Val Arg Gln Ala Ile Gly Lys Ala Ile Val Ala Tyr Tyr 80 85 90 GCT AAG AAC GTC GAT GCC GCC TCT GCC CTC GAG ATC AAG AAG GCT CTC 338 Ala Lys Asn Val Asp Ala Ala Ser Ala Leu Glu Ile Lys Lys Ala Leu 95 100 105 GTC GCC TAC GAC CGA ACC CTC CTC ATC GCC GAT CCC CGA CGA ATG GAG 386 Val Ala Tyr Asp Arg Thr Leu Leu Ile Ala Asp Pro Arg Arg Met Glu 110 115 120 CCC AAG AAG TTC GGA GGA CCC GGA GCC CGA GCC CGA GTC CAG AAG TCT 434 Pro Lys Lys Phe Gly Gly Pro Gly Ala Arg Ala Arg Val Gln Lys Ser 125 130 135 TAC CGA TAAAAAGTGT TTGTCTTGTG GTCTGGCGGG TCATCTATCC AACATCTTTG 490 Tyr Arg 140 GAAAANANTT GTTTGGGTCA TATGTCATGC CTCTTTATGG AAAAAAAAAA AA 542 141 amino acids amino acid linear protein not provided 51 Met Ser Val Ala Val Gln Thr Phe Gly Lys Lys Lys Thr Ala Thr Ala 1 5 10 15 Val Ala His Ala Thr Pro Gly Arg Gly Leu Ile Arg Leu Asn Gly Gln 20 25 30 Pro Ile Ser Leu Ala Glu Pro Ala Leu Leu Arg Tyr Lys Tyr Tyr Glu 35 40 45 Pro Ile Leu Val Ile Gly Ala Glu Lys Ile Asn Gln Ile Asp Ile Arg 50 55 60 Leu Lys Val Lys Gly Gly Gly His Val Ser Gln Val Tyr Ala Val Arg 65 70 75 80 Gln Ala Ile Gly Lys Ala Ile Val Ala Tyr Tyr Ala Lys Asn Val Asp 85 90 95 Ala Ala Ser Ala Leu Glu Ile Lys Lys Ala Leu Val Ala Tyr Asp Arg 100 105 110 Thr Leu Leu Ile Ala Asp Pro Arg Arg Met Glu Pro Lys Lys Phe Gly 115 120 125 Gly Pro Gly Ala Arg Ala Arg Val Gln Lys Ser Tyr Arg 130 135 140 

What is claimed is:
 1. Recombinant DNA comprising a transcription promoter and a downstream sequence to be expressed, in operable inkage therewith, wherein the transcription promoter comprises a region found upstream of the open readings frame of a highly expressed Phaffia gene is capable of making Phaffia rhodozyma transformed with a DNA construct having said promoter linked up front of the G418 resistance marker resistant to G-418 in concentrations exceeding 200 μg per liter culture medium, wherein said highly expressed Phaffia gene is a glyceraldehyde-3-phosphate dehydrogenase gene or a ribosomal protein encoding gene.
 2. A microorganism harboring a recombinant DNA according to claim
 1. 3. A microorganism according to claim 2, which is Phaffia rhodozyma.
 4. A microorganism according to claim 3, having the recombinant DNA integrated into its genome in an amount of 50 copies or more.
 5. Recombinant DNA of claim 1 wherein said open reading frame encodes one of the amino acid sequences depicted in any one of SEQ ID NOs: 24 to
 50. 6. A recombinant DNA according to claim 1 wherein said downstream sequence to be expressed is heterologous with respect to the transcription promoter sequence.
 7. A recombinant DNA according to claim 1, wherein the downstream sequence encodes a polypeptide responsible for reduced sensitivity against a selective agent.
 8. A recombinant DNA according to claim 7, wherein said selective agent is G418.
 9. A recombinant DNA according to claim 1, wherein the said downstream sequence to be expressed encodes an enzyme involved in the carotenoid biosynthesis pathway.
 10. A recombinant DNA according to claim 9, wherein said downstream sequence to be expressed encodes an enzyme having an activity selected from the group consisting of isopentenyl pyrophosphate isomerase, geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and lycopene cyclase.
 11. A recombinant DNA according to claim 10, wherein said downstream sequence to be expressed encodes an enzyme having an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO:
 23. 12. A recombinant DNA according to claim 1 wherein said recombinant DNA comprises further a transcription terminator downstream from the said DNA sequence to be expressed, in operable linkage therewith.
 13. A recombinant DNA according to claim 12, wherein the terminator is a GAPDH-encoding gene terminator fragment.
 14. A recombinant DNA according to claim 1 wherein the recombinant DNA is in the form of a vector capable of replication and/or integration in a host organism.
 15. A recombinant DNA according to claim 14, further comprising DNA transcribed into Phaffia ribosomal RNA.
 16. A recombinant DNA according to claim 15, which is linearized by cleaving inside the DNA transcribed into Phaffia ribosomal RNA.
 17. A method for obtaining a transformed Phaffia strain, comprising the steps of (a) contacting cells or protoplasts of a Phaffia strain with the recombinant DNA of claim 1 under conditions conducive to uptake thereof, and (b) identifying Phaffia rhodozyma cells or protoplasts having obtained the said recombinant DNA in expressible form.
 18. A method according to claim 17, comprising the additional step of providing an electropulse after contacting of Phaffia cells or protoplasts with the said recombinant DNA.
 19. A transformed Phaffia strain obtainable by a method according to claim 17 wherein said strain, upon cultivation, expresses expression of the said downstream sequence.
 20. A transformed Phaffia strain according to claim 19, wherein the said downstream sequence codes for a pharmaceutical protein.
 21. A method for producing a pharmaceutical protein which method comprises culturing a transformed Phaffia strain according to claim 20 under conditions conducive to the production of the said protein.
 22. A transformed Phaffia strain according to claim 19 wherein the said Phaffia strain contains at least 10 copies of the said recombinant DNA integrated into its genome.
 23. An isolated DNA fragment comprising a Phaffia GAPDH-gene, or a functional fragment thereof including a promoter which is capable of making Phaffia rhodozyma transformed with a DNA construct having said promoter linked up front of the G418 resistance marker resistant to G-418 in concentrations exceeding 200 μg per liter culture medium.
 24. A method to prepare a recombinant DNA construct which comprises ligating a functional fragment according to claim 23 into said construct.
 25. The method according to claim 24, wherein said fragment is a regulatory region normally located upstream or downstream of the open reading frame coding for GAPDH in Phaffia rhodozyma.
 26. A transformed Phaffia rhodozyma strain which overexpresses a nucleotide sequence encoding an enzyme involved in the carotenoid biosynthesis pathway, wherein the nucleotide sequence is operably linked to a promoter which is capable of making Phaffia rhodozyma transformed with a DNA construct having said promoter linked up front of the G418 resistance marker resistant to G-418 in concentrations exceeding 200 μg per liter culture medium.
 27. A transformed Phaffia rhodozyma strain according to claim 26, which produces inreased amounts of astaxanthin relative to its untransformed ancestor.
 28. An isolated DNA comprising a nucleotide sequence encoding an enzyme that has an activity in the carotenoid biosynthetic pathway operably linked to a promoter which is capable of making Phaffia rhodozyma transformed with a DNA construct having said promoter linked up front of the G418 resistance marker resistant to G-418 in concentrations exceeding 200 μg per liter culture medium.
 29. An isolated DNA according to claim 28, wherein said activity is selected from isopentenyl pyrophosphate isomerase activity, geranylgeranyl pyrophosphate synthase activity, phytoene synthase activity, phytoene desaturase activity and lycopene cyclase activity.
 30. The isolated DNA of claim 28 wherein said enzyme has an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO 17, SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO:
 23. 31. The isolated DNA of claim 28, wherein the carotenoid biosynthetic pathway is one from Phaffia rhodozyma.
 32. Recombinant DNA comprising the encoding nucleotide sequence according to claim
 28. 33. Recombinant DNA according to claim 32 being in the form of a vector.
 34. Recombinant DNA according to claim 32, wherein said encoding nucleotide sequence is operably linked to a transcription promoter which is expressed in a suitable host, said isolated DNA sequence optionally being linked to a transcription terminator functional in the said host.
 35. Recombinant DNA according to claim 34, wherein said host is a Phaffia strain.
 36. Recombinant DNA according to claim 34 wherein the transcription promoter is from a glycolytic pathway gene present in Phaffia.
 37. Recombinant DNA according to claim 36, wherein said glycolytic pathway gene is a gene coding for Glyceraldehyde-3-Phosphate Dehydrogenase.
 38. Recombinant DNA according to claim 34 wherein the transcription promoter is from a ribosomal protein encoding gene.
 39. Recombinant DNA according to claim 34 wherein the transcription promoter comprises a region found upstream of the open reading frame encoding a protein as represented by one of the amino acid sequences depicted in any one of SEQ ID NOs: 24 to
 50. 40. Recombinant DNA according to claim 34 wherein said recombinant DNA comprises further a transcription terminator downstream from the said encoding nucleotide sequence to be expressed, in operable linkage therewith, which terminator is a Phaffia transcription terminator.
 41. A host modified to contain the recombinant DNA according to claim
 32. 42. A host according to claim 41, which is a Phaffia strain.
 43. A method for producing an enzyme involved in the carotenoid biosynthesis pathway, which method comprises culturing a host according to claim 41 under conditions conducive to the production of said enzyme.
 44. A method for producing a carotenoid which method comprises cultivating a host according to claim 41 under conditions conducive to the production of a carotenoid.
 45. A method according to claim 44, wherein the carotenoid is astaxanthin. 